Optimal Antitumor Efficacy Research Articles

Exploration and application of treating OSCC by a precisely concentrated synergistic agent in combination with thermally responsive release of effective therapeutic dosages of cisplatin

Platinum-based drugs, foremost among them Cisplatin (CDDP), currently constitute the primary line of chemotherapy for the treatment of oral squamous cell carcinoma (OSCC). However, the extensive utilization of these drugs often results in undesirable systemic toxic side effects. Consequently, in clinical practice, emphasis has shifted towards the adoption of combination drug strategies, aimed at minimizing the dosage of platinum-based drugs while preserving optimal anti-tumor efficacy. Prior research has concentrated on the co-loading of drugs with synergistic potential into a unified nanosystem, yet this approach does not guarantee that the drugs will remain within the concentration range necessary for synergistic effects following nanocarrier release, thus limiting the full exploitation of their synergistic anti-tumor properties. This study has pioneered the discovery that the HSP90 inhibitor 17AAG exhibits remarkable synergistic effects with CDDP in the treatment of OSCC. Importantly, 17AAG can allow CDDP to still exert effective tumor killing effects at lower concentrations. Furthermore, specific concentrations of 17AAG demonstrate synergistic effects with a wide range of CDDP concentrations, positioning it as a promising candidate for the sustained synergistic augmentation of CDDP released from nanocarriers. Additionally, we have augmented CDDP targeting through the employment of a hybrid membrane strategy, encapsulating CDDP and photosensitizers concurrently. This approach enhances CDDP’s targeting precision and enables responsive tracking and release. Notably, we have observed that altering the tumor cell membrane type significantly modifies the tumor targeting profile of the hybrid membrane, suggesting that cell membrane-modified nanocarriers have potential value in achieving personalized tumor therapy. In summary, we have formulated a novel CDDP-based targeted therapy strategy for OSCC, which holds promising implications for future treatment of this malignancy.

A carrier free delivery system of a MAGL inhibitor is effective on ovarian cancer

Monoacylglycerol lipase (MAGL) is a promising target for cancer therapy due to its involvement in lipid metabolism and its impact on cancer hallmarks like cell proliferation, migration, and tumor progression. A potent reversible MAGL inhibitor, MAGL23, has been recently developed by our group, demonstrating promising anticancer activities. To enhance its pharmacological properties, a nanoformulation using nanocrystals coated with albumin was prepared (MAGL23AF). In a previous work, the formulated inhibitor showed potency in ovarian and colon cancer cell lines in terms of IC50, and was tested on mice in order to assess its biocompatibility, organs biodistribution and toxicity. In the present work, we expanded the investigation to assess the potential in vivo application of MAGL23AF. Stability assays in serum and in human derived microsomes showed a good structural stability in physiological conditions of MAGL23AF. The antitumor efficacy tested on mice bearing ovarian cancer tumor xenografts demonstrated that MAGL23AF is more potent than the non-formulated drug, leading to necrosis-driven cancer cell death. In vivo studies revealed that albumin-complexed nanocrystals improved the therapeutic window of MAGL23, exhibiting a favorable biodistribution with slightly increased accumulation in the tumor. In conclusion, the MAGL23AF showed increased in vitro stability in conditions mirroring the bloodstream environment and hepatic metabolism coupled with an optimal antitumor efficacy in vivo. These results not only validates the efficacy of our formulation but also positions it as a promising strategy for addressing challenges related to the solubility of drugs in body fluids.

Preclinical Study of DZD8586, a Non-Covalent LYN/BTK Dual Inhibitor with Excellent BBB Penetration, for the Treatment of B-Cell Non-Hodgkin Lymphoma (B-NHL)

Introduction Bruton's Tyrosine Kinase (BTK) inhibitors have been approved for the treatment of B-NHL, such as CLL and MCL. However, resistance to BTK inhibitors still inevitably occurred, and different types of mutations were identified post covalent and non-covalent BTK inhibitor treatment. Although BTK inhibitors showed some activities in the treatment of relapsed or refractory non-germinal center B-cell (non-GCB) DLBCL, no drugs have been approved due to unsustainable tumor response. It is hypothesized that blockage of BTK pathway alone is not sufficient to achieve optimal anti-tumor efficacy in DLBCL. Due to limited blood-brain barrier (BBB) penetration of the current therapies, treatment of central nervous system lymphoma (CNSL) remains a clinical challenge. DZD8586 is a rationally designed, oral, non-covalent, LYN and BTK dual inhibitor with excellent BBB penetration as a potential treatment option for B-NHL. Here we report the preclinical activity of DZD8586 in lymphoma cell lines and animal models which supports its clinical development to address the above unmet medical needs. Methods The enzymatic activity of DZD8586 against multiple kinases was assessed at Eurofins panel with purified enzymes. TMD-8-IbruR cells carrying the C481S mutation were generated by stepwise induction of increasing concentrations of Ibrutinib. Cells carrying pirtobrutinib resistance mutations were obtained by transfection of different BTK mutations into RI-1 cells using lentivirus. Cell growth inhibition was determined by CellTiter-Glo. A pBTK enzyme-linked immunosorbent assay (ELISA) was used to evaluate the pharmacodynamic profile of DZD8586. Western blot was used to test the modulations of signaling pathways with DZD8586 treatment. Animal models were established by subcutaneous and intracranial inoculation of tumor cells, respectively, and animals were treated with DZD8586 or other BTK inhibitors. The anti-tumor efficacy was assessed by comparing the change in mean tumor volume between the different treatment groups and vehicle controls. Results Kinase panel profiling showed that DZD8586 potently inhibited LYN and BTK, with good selectivity against other kinases. In cell lines expressing C481X and pirtobrutinib resistance mutations, DZD8586 demonstrated concentration dependent anti-proliferative effects, with similar GI 50s among cells carrying different BTK mutations. In a panel of DLBCL cell lines, DZD8586 exhibited potent cell growth inhibition and induced cell death in a variety of cell lines, and its effect correlated with modulations of both LYN and BTK pathways. In both subcutaneous and CNS tumor models, DZD8586 exhibited profound tumor growth inhibition in a dose-dependent manner, and induced tumor regression at the doses of 50 mg/kg and above. There was a good correlation between plasma concentrations of DZD8586 and modulations of pBTK in whole blood and tumor tissue. DZD8586 has good cell permeability and is not significantly transported by P-gp and BCRP expressed at the BBB. The K puu,CSF of DZD8586 in rat and monkey were 1.2 and 1.3, respectively, suggesting its excellent BBB penetration in humans. Conclusion DZD8586 is a novel non-covalent LYN/BTK dual inhibitor, which could overcome resistance mutations to the approved covalent and non-covalent BTK inhibitors, and derive clinical benefit to patients with DLBCL and CNSL by blocking both BTK-dependent and BTK-independent signaling pathways with excellent BBB penetration.

Universal chimeric Fcγ receptor T cells with appropriate affinity for IgG1 antibody exhibit optimal antitumor efficacy.

Developing universal CARs with improved flexible targeting and controllable activities is urgently needed. While several studies have suggested the potential of CD16a in tandem with monoclonal antibodies to construct universal CAR-T cells, the weak affinity between them is one of the limiting factors for efficacy. Herein, we systematically investigated the impact of Fcγ receptor (FcγR) affinity on CAR-T cells properties by constructing universal CARs using Fcγ receptors with different affinities for IgG1 antibodies, namely CD16a, CD32a, and CD64. We demonstrated that the activities of these universal CAR-T cells on tumor cells could be redirected and regulated by IgG1 antibodies. In xenografted mice, 64CAR chimeric Jurkat cells with the highest affinity showed significant antitumor effects in combination with herceptin in the HER2 low expression U251 MG model. However, in the CD20 high expression Raji model, 64CAR caused excessive activation of CAR-T cells, which resulted in cytokine release syndrome (CRS) and the decline of antitumor activity, and 32CAR with a moderate affinity brought the best efficacy. Our work extended the knowledge about FcγR-based universal CAR-T cells and suggested that only the FcγRCAR with an appropriate affinity can offer the optimal antitumor advantages of CAR-T cells.

Nanomicellar Formulations Loaded with Histamine and Paclitaxel as a New Strategy to Improve Chemotherapy for Breast Cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Currently, paclitaxel (PTX) represents the first-line therapy for TNBC; however it presents a hydrophobic behavior and produces severe adverse effects. The aim of this work is to improve the therapeutic index of PTX through the design and characterization of novel nanomicellar polymeric formulations composed of a biocompatible copolymer Soluplus® (S), surface-decorated with glucose (GS), and co-loaded either with histamine (HA, 5 mg/mL) and/or PTX (4 mg/mL). Their micellar size, evaluated by dynamic light scattering, showed a hydrodynamic diameter between 70 and 90 nm for loaded nanoformulations with a unimodal size distribution. Cytotoxicity and apoptosis assays were performed to assess their efficacy in vitro in human MDA-MB-231 and murine 4T1 TNBC cells rendering optimal antitumor efficacy in both cell lines for the nanoformulations with both drugs. In a model of TNBC developed in BALB/c mice with 4T1 cells, we found that all loaded micellar systems reduced tumor volume and that both HA and HA-PTX-loaded SG micelles reduced tumor weight and neovascularization compared with the empty micelles. We conclude that HA-PTX co-loaded micelles in addition to HA-loaded formulations present promising potential as nano-drug delivery systems for cancer chemotherapy.

Antitumor Immunotherapy of Sialic Acid and/or GM1 Modified Coenzyme Q10 Submicron Emulsion.

Immunotherapy is a novel therapeutic approach for controlling and killing tumor cells by stimulating or reconstituting the immune system, among which T cells serve as immune targets. Herein, we used coenzyme Q10 (CoQ10), which has both immune activation and avoids adverse reactions, as a model drug and developed four CoQ10 submicron emulsions modified with sialic acid (SA) and/or monosialotetrahexosyl ganglioside (GM1). On the one hand, SA interacts with L-selectins on the surface of T cells after entering the circulatory system, leading to activation of T cells and enhancement of antitumor immune responses. On the other hand, owing to its immune camouflage, GM1 can prolong the circulation time of the preparation in the body, thereby increasing the accumulation of the drug at the tumor site. In vitro and in vivo experiments showed that SA-modified preparations exhibited stronger immune activation and inhibition of tumor proliferation. Pharmacokinetic experiments showed that GM1-modified preparations have longer circulation times in vivo. However, SA and GM1 co-modification did not produce a synergistic effect on the preparation. In conclusion, the SA-modified CoQ10 submicron emulsion (Q10-SE) showed optimal antitumor efficacy when administered at a medium dose (6mg CoQ10 kg-1). In this study, the submicron emulsion model was used as a carrier, and the tumor-bearing mice were used as animal models. In addition, CoQ10 submicron emulsion was modified with SA-CH with active targeting function and/or GM1 with long-circulation function to explore the antitumor effects of different doses of CoQ10 submicron emulsion, and to screen the best tumor immunotherapy formulations of CoQ10.

The Role of Neutrophils in Oncolytic Orf Virus-Mediated Cancer Immunotherapy.

Neutrophils are innate leukocytes with diverse effector functions that allow them to respond to pathogens rapidly. Accumulating evidence has highlighted these cells’ complex roles in the host’s response to viral infections and tumor progression. Oncolytic virotherapy is emerging as a promising treatment modality in the armamentarium of cancer therapeutics. Oncolytic viruses preferentially kill cancer cells and stimulate tumor-associated inflammation, resulting in tumor regression. Assessing the activity of individual effector cell subsets following oncolytic virotherapy is important in identifying their contribution to antitumor immunity. In this study, we investigated the role of neutrophils in oncolytic Orf-virus-mediated immunotherapy in a murine model of pulmonary melanoma metastases. The systemic administration of the Orf virus stimulated a dramatic increase in the number of leukocytes in circulation and within the tumor microenvironment, most of which were neutrophils. Analysis of tumor-burdened lungs shortly after therapy revealed significant numbers of phenotypically immature neutrophils, with the enhanced expression of molecules affiliated with activation, migration, and cytotoxicity. Neutrophils stimulated by Orf virus therapy were directly tumoricidal through tumor necrosis factor-α-mediated effects and were required for optimal antitumor efficacy following Orf virus therapy. Taken together, these data reveal neutrophils as a crucial innate effector to consider when investigating oncolytic virotherapy.

A 2D Nanoradiosensitizer Enhances Radiotherapy and Delivers STING Agonists to Potentiate Cancer Immunotherapy.

Despite potent preclinical antitumor activity, activation of stimulator of interferon genes (STING) has shown modest therapeutic effects in clinical studies. Many STING agonists, including 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), show poor pharmacokinetic properties for sustaining STING activation in tumors and achieving optimal antitumor efficacy. Improved delivery of STING agonists and their effective combination with other treatments are needed to enhance their therapeutic effects. Herein, a 2D nanoplatform, cGAMP/MOL, is reported via conjugating cGAMP to a nanoscale metal-organic layer (MOL) for simultaneous STING activation and radiosensitization. The MOL not only exhibits strong radiosensitization effects for enhanced cancer killing and induction of immunogenic cell death, but also retains cGAMP in tumors for sustained STING activation. Compared to free cGAMP, cGAMP/MOL elicits stronger STING activation and regresses local tumors upon X-ray irradiation. Further combination with an immune checkpoint inhibitor bridges innate and adaptive immune systems by activating the tumor microenvironment to elicit systemic antitumor responses.

A Physiologically Based Pharmacokinetic Framework for Quantifying Antibody Distribution Gradients from Tumors to Tumor-Draining Lymph Nodes.

Immune checkpoint blockades prescribed in the neoadjuvant setting are now under active investigation for many types of tumors, and many have shown early success. The primary tumor (PT) and tumor-draining lymph node (TDLN) immune factors, along with adequate therapeutic antibody distributions to the PT and TDLN, are critical for optimal immune activation and anti-tumor efficacy in neoadjuvant immunotherapy. However, it remains largely unknown how much of the antibody can be distributed into the PT-TDLN axis at different clinical scenarios. The goal of the current work is to build a physiologically based pharmacokinetic (PBPK) model framework capable of characterizing antibody distribution gradients in the PT-TDLN axis across various clinical and pathophysiological scenarios. The model was calibrated using clinical data from immuno-PET antibody-imaging studies quantifying antibody pharmacokinetics (PK) in the blood, PTs, and TDLNs. The effects of metastatic lesion location, tumor-induced compression, and inflammation, as well as surgery, on antibody concentration gradients in the PT-TDLN axis were characterized. The PBPK model serves as a valuable tool to predict antibody exposures in various types of tumors, metastases, and the associated lymph node, supporting effective immunotherapy.

Preclinical Results of an Allogeneic, Universal CD19/CD7-Targeting CAR-T Cell Therapy (GC502) for B Cell Malignancies

BackgroundAutologous CD19 CAR-T therapies show very promising clinical efficacy, but are limited in their applicability by several factors including cost, time to manufacture, and other factors involving patients own T-cell qualities. GC027, a CD7 targeting allogeneic, universal CAR-T (UCAR-T) currently in development for the treatment of T-cell acute lymphoblastic leukemia (T-ALL) has demonstrated robust expansion and anti-leukemia efficacy with a manageable safety profile in an investigator-initiated trial in China. These data suggest that, a single CD7 targeting CAR-T therapy is able to generate a therapeutic window by suppressing host vs graft (HvG) rejection of UCAR-T cells by patients' own NK and T cells, and achieve efficacy in patients with T-ALL. Based on these findings we developed GC502, a CD19/CD7 dual-targeting, allogeneic CAR-T therapy for B-cell malignancies, in which the CD19 CAR moiety targets malignant cells while CD7 CAR moiety suppresses HvG in variety of preclinical models.MethodsGC502 was manufactured using leukopaks from HLA-unmatched healthy donors. It contains a 4-1BB based 2 nd-generation dual targeting CAR comprising an anti-CD19 and an anti-CD7 single-chain variable fragments (scFvs). TRAC and CD7 loci were disrupted to avoid graft vs host disease and fratricide, respectively. To select the leading CAR candidate, CAR expression and functionalities of CAR constructs with different heavy-light (H-L) chain orientations of the dual CAR were analyzed via in vitro assays and mouse xenograft models, in comparing to single CD19 CAR and CD7 CAR products. To achieve optimal anti-tumor efficacy, a T-cell enhancer was included in the CAR construct.ResultGene editing and dual CAR orientation selectionTRAC and CD7 double knockout efficiencies were constantly above 97% across multiple donor pan T cells. Although CD19/CD7 CAR expression levels in different H-L chain orientations were similar, in the final CAR-T product as measured by flow cytometry (FCM) analysis, significant difference was observed in their cytotoxicity and in vitro expansion under repeated antigen stimulations by CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cell line Nalm6 and CD7+ T-cell line CCRF-CEM. CAR candidates mediated the strongest cytotoxicity and most durable response were selected for further optimizations.CAR construct optimizationsFor the leading candidates, we first assessed the dual CAR efficacy after incorporation of an enhancer. While the IL-2, TNFα and IFNγ secretion levels were comparable, enhancer addition significantly improved tumor killing and CAR-T cell expansion under repeated stimulations by either CD19+ or CD7+ target cells. Anti-leukemia response under sub-optimal CAR-T cell dosages were also greatly enhanced as assessed by both B-ALL and T-ALL mouse xenograft models.GC502 CAR functionality comparison to single CAR products with proven clinical efficaciesGC502 and GC027 were compared for their CD7 CAR function to assess their anti-HvG activities. GC502 and GC027 exhibited comparable toxicities towards pan T cells and similar efficacies in a highly malignant T-ALL mouse model. The CD19 CAR functionality of GC502 were evaluated and compared to a 2 nd generation CD19 CAR product comprising a FMC63 scFv and a 4-1BB-CD3ȥ signaling domain. In a Raji based B-ALL mouse xenograft model, both products rapidly eliminated cancer cells. While CD19 CAR treated mice showed signs of relapse at 2 weeks post CAR-T infusion, GC502 treatment group maintained “leukemia free” status till the end of study (Day28).ConclusionGC502 was optimized for CD19/CD7 dual CAR functionality and in vivo durability. It demonstrated robust anti-tumor efficacy and promising potentials to suppress HvG. This report presents an example that the dual CAR design of GC502 may serve as a novel “off-the-shelf” CAR-T technology. DisclosuresGe: GracellBiotechnologies Ltd: Current Employment, Current equity holder in publicly-traded company. Yang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sun: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Chen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Qiu: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Yin: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Shen: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Sersch: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Cao: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company. Wang: GracellBiotechnologies Inc: Current Employment, Current equity holder in publicly-traded company.

Photo-Driven Delivery of 125I-Labeled Nanomicelles for Nucleus-Targeted Internal Conversion Electron-Based Cancer Therapy.

As a kind of high linear energy transfer (LET) radiation, internal conversion electrons are emitted from some radionuclides, such as 125I, triggering severe DNA damage to tumor cells when transported into the nucleus. Herein, we develop a curcumin-loaded nanomicelle composed of a photosensitizer chlorin e6 (Ce6) and amphiphilic poly(ethylene glycol) (poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) (C18-PMH-PEG)) to deliver 125I into the nucleus under 660 nm laser irradiation, leading to the optimized imaging-guided internal conversion electron therapy of cancer. Ce6-containing nanomicelles (Ce6-C18-PEG) self-assemble with nucleus-targeted curcumin (Cur), obtaining Ce6-C18-PEG/Cur nanoparticles. After labeling Cur with 125I, Ce6-C18-PEG/Cur enables single-photon emission computed tomography and fluorescence imaging of the tumor, serving as a guide for follow-up laser irradiation. Notably, the 660 nm laser-triggered photodynamic reaction of Ce6 optimizes the delivery of Ce6-C18-PEG/125I-Cur at various stages, including tumor accumulation, cellular uptake, and lysosome escape, causing plenty of 125I-Cur to enter the nucleus. By this strategy, Ce6-C18-PEG/125I-Cur showed optimal antitumor efficacy and high biosafety in mice treated with local 660 nm laser irradiation using efficient energy deposition of internally converted electrons over short distances. Therefore, our work provides a novel strategy to optimize 125I delivery for tumor treatment.

Multifunctional Microspheres Dual-Loaded with Doxorubicin and Sodium Bicarbonate Nanoparticles to Introduce Synergistic Trimodal Interventional Therapy.

Lactic acid in the tumor microenvironment is highly correlated with the prognosis of tumor chemoembolization, but there are limited clinical strategies to deal with it. To improve the efficacy, NaHCO3 nanoparticles are innovatively introduced into drug-loaded microspheres to neutralize lactic acid in the tumor microenvironment. Here we showed that multifunctional ethyl cellulose microspheres dual-loaded with doxorubicin (DOX) and NaHCO3 nanoparticles (DOX/NaHCO3-MS) presented excellent antitumor effects by improving the pH of the tumor microenvironment. The homeostasis of the tumor microenvironment was continuously disturbed due to the sustained release of NaHCO3 nanoparticles, which also led to a significant increase in tumor cell apoptosis (compared with the control and DOX-MS groups). We also showed that the administration of DOX/NaHCO3-MS via the hepatic artery in a rabbit model of VX2 orthotopic liver cancer resulted in optimal antitumor efficacy, and the area of tumor necrosis at the embolization site was significantly increased and the proliferation of tumor cells was significantly weakened. The designed DOX/NaHCO3-MS exhibited strong synergistic antitumor effects of embolization, chemotherapy, and tumor microenvironment improvement. The present microspheres provided a strategy for the enhancement of the chemoembolization of hepatocellular carcinoma, which could also be extended to other clinical embolization treatments for blood-rich solid tumors.

Doxorubicin@ PEPylated interferon-polydisulfide: A multi-responsive nanoparticle for enhanced chemo–protein combination therapy

The combination of chemotherapy and protein therapy holds immense promise for tumor treatment. However, conventional bolus formulation or simple co-administration fails to achieve maximized efficacy due to the distinct physiological and pharmacological properties of small molecular drugs and protein therapeutics. As such, developing a co-delivery system for optimal antitumor efficacy remains a great challenge. Herein, we achieve the synergistic co-delivery of human interferon-α2b (IFN) and doxorubicin (Dox) by the facile synthesis of a multifunctional and stimuli-responsive protein-drug-polymer (PDP) conjugate IFN-PolyDox-PEP. IFN-PolyDox-PEP is constituted of IFN, a polydisulfide tethering multiple copies of Dox (PolyDox), and a stealthy polypeptide (PEP) for long circulation. IFN-PolyDox-PEP self-assembles into nanoparticles of 122 nm in diameter and is able to release its therapeutic cargos in response to tumor microenvironment cues such as matrix metalloproteinase, acidic pH, and reducing glutathione. The in vivo synergistic effect of IFN-PolyDox-PEP is demonstrated by the superior antitumor efficacy as compared with the co-administration of IFN-polypeptide conjugate and free Dox. This work demonstrates the power of highly efficient chemistry in constructing well-defined PDP conjugates, which are promising hybrid macromolecules for various chemo-protein combination therapies.

Abstract 4508: Modulating the release rate of polyethylene glycol (PEG) polymers from interleukin-2 (IL-2) PEG conjugates to optimize anti-tumor efficacy

Abstract The cytokine interleukin-2 (IL-2) is an endogenous agonist of the IL-2 pathway that activates and expands tumor killing lymphocytes. High doses of IL-2 are needed to activate CD8 T cells and NK cells, through binding on low-affinity IL-2 receptor beta gamma subunits (IL-2Rβγ). Severe toxic side effects associated with high doses, as well as the poor PK profile of IL-2, have limited its clinical usage. In addition of the toxicities, IL-2 also stimulates proliferation and activation of regulatory T cells (Tregs) through binding on the high-affinity heterotrimeric IL-2 receptor alpha beta gamma subunits (IL-2Rαβγ), which may antagonize the intended anti-tumor immunity. We report on an IL-2 PEGylation strategy to improve the pharmacokinetic properties of IL-2. Utilizing our proprietary PEGylation technology, IL-2 was covalently connected with multiple polyethylene glycol (PEG) polymers through releasable linkers. Our technology enabled the conjugation of IL-2 with average of 5-6 polyethylene glycol (PEG) polymers in high efficiency and 80-90% isolated yield. Anti-tumor efficacy of IL-2 was optimized by modulating the hydrolysis rate of the releasable linkers connecting IL-2 and polymers. The releasable linkers used in the study had the hydrolysis rate in the range of 15 to 120 hours to release one PEG polymer from the conjugates. The conjugates with hydrolysis rate of 20-60 h showed the optimal anti-tumor efficacy in B16F10 animal model. These results suggest that our novel approach of IL-2 PEGylation may optimize IL-2 biological activity and anti-tumor efficacy. Modulating the releasable rate of the PEG polymer would optimize the pharmacokinetic properties of the released active IL-2 species and enable the optimized anti-tumor efficacy. We are currently exploring the usage of our technology platform to further engineer IL-2 as a non-IL2Rα binding conjugate for improved toxicity profile. Citation Format: Hui Li, Yuntao Song, Haiping Zhou, Chuan Liao, Meng Xu, Chuang Li. Modulating the release rate of polyethylene glycol (PEG) polymers from interleukin-2 (IL-2) PEG conjugates to optimize anti-tumor efficacy [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 4508.

Abstract 5345: Heterogeneous tumor PSMA expression represents a resistance mechanism to PSMA-targeted radioligand therapy

Abstract Radioligand therapy (RLT) with prostate-specific membrane antigen (PSMA)-targeting ligands is effective in ~50% of patients with advanced prostate cancer (PC). Causes for RLT failure are not well understood. The prevalent PSMA heterogeneity in PC might contribute to RLT failure. Here we investigate the relationship between RLT efficacy and PSMA levels per cell, and PSMA heterogeneity. PC cells expressing different levels of PSMA (RM1-PSMA low, medium, high, or RM1-YFP that do not express PSMA), or a mix of PSMA− and PSMA+ cells (RM1-YFP/ RM1-PSMA-high; PC3/ PC3-PIP) at various ratios were subcutaneously injected into NSG mice. Mice were treated with 177Lu- or 225Ac-PSMA617 (RLT) and tumor growth was monitored. In a subset of mice, radioligand uptake (γ-counting), DNA damage and PSMA expression (anti-53BP1 and -PSMA immunohistochemistry, respectively) were quantified in tumors resected 2 days after RLT. Increasing PSMA levels and fractions of PSMA+ cells improved RLT efficacy in both, the RM1 and PC3 model. PSMA expression correlated with radioligand uptake into the tumor and the degree of DNA damage and. Treatment with 225Ac-PSMA617 (vs. 177Lu-PSMA617) improved RLT outcomes and tended to enhance the differences in therapeutic efficacy between experimental groups. Taken together, we demonstrate in mouse models of PC that optimal anti-tumor efficacy of RLT hinges on homogenously high target expression. Although PSMA-RLT is effective even in tumors with low PSMA levels or with a small number of PSMA+ cells, low or heterogeneous PSMA expression might result in undertreatment and selection of treatment resistant clones. Systematic assessment of intra- and inter-lesion PSMA heterogeneity is currently not feasible clinically; however, this issue might be addressed by individual patient dosimetry to optimize safely delivered maximal tumor doses. Clinical studies designed to determine intra- and inter-lesion PSMA heterogeneity and to optimize PSMA-RLT for each patient are highly warranted. Citation Format: Katharina Lueckerath, Kyle Current, Catherine Meyer, Clara Magyar, Christine E. Mona, Joel Almajano, Roger Slavik, Andreea D. Stuparu, Chloe Cheng, David Dawson, Caius Radu, Johannes Czernin. Heterogeneous tumor PSMA expression represents a resistance mechanism to PSMA-targeted radioligand therapy [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 5345.

Targeting innate sensing in the tumor microenvironment to improve immunotherapy.

The innate immune sensing pathways play critical roles in the defense against pathogen infection, but their roles in cancer immunosurveillance and cancer therapies are less defined. We propose that defective innate immune sensing inside the tumor microenvironment might limit T-cell responses to immunotherapy. A recent mechanistic understanding of conventional therapies revealed that both innate immune sensing and T-cell responses are essential for optimal antitumor efficacy. T-cell-based immunotherapy, particularly immune checkpoint blockade, has achieved great success in reactivating antitumor immune responses to lead to tumor regression, but only in a small fraction of patients. Therefore, incorporating conventional therapy that can increase innate sensing and immunotherapy should lead to promising strategies for cancer patients. Here, we review the innate sensing pathways related to cancer initiation/progression and therapies, summarize the recent key findings in innate immune sensing related to conventional therapies, evaluate current combination strategies, and highlight the potential issues of combinational therapies in terms of antitumor efficacy and toxicities.

Integrated profiling of T cells and tumor cells demonstrates metabolic adaptation essential for melanoma cell therapy

Abstract T cell-based therapies have shown promising results, but adoptive T cell therapy (ACT) for solid tumors like melanoma treatment has met limited success. The goal of this study was to identify attributes of T cell fitness important for optimal anti-tumor efficacy. This was accomplished by integrated profiling of expanded tumor infiltrating lymphocytes, TILs (responders (CR) and progressive diseases (PD)) co-cultured overnight with their autologous primary tumor cells with the aid of a suite of single-cell, transcriptional, proteomic and functional assays to construct a genome-scale metabolic model of the metabolism of TILs and tumor cells in direct competition with each other. Sorted live TILs after co-culture were used to performed RNA-seq, and proteome-wide profiling by mass spectrometry. Gene enrichment of the CR TIL suggested an increased expression in genes involving processes such as glycolysis, hypoxia, adipogenesis, and mTORC1 signaling. PD tumors showed an increase for epithelial to mesenchymal transition (EMT) and glycolysis. Comparisons of transcripts and proteins in both CR and PD were tightly correlated (Spearman rank 0.765). Since patient-derived cell numbers were limited (<50,000), we utilized genome-scale metabolic models to infer relevant metabolic pathways by comparison to the human metabolic Atlas (HMR2). We observed high enrichment in fatty acid β-oxidation processes in different organelles related to CR TIL and tumor. Lastly, we examined if increasing fatty acid oxidation in TILs might enable their increased survival in metabolically replete environments. These results show that the fatty acid metabolism pathway is associated with the ACT efficacy, and it can contribute to improving treatment.

Role of tumor-associated neutrophils in regulation of tumor growth in lung cancer development: A mathematical model.

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

Regulatory Mechanism of Localized Surface Plasmon Resonance Based on Gold Nanoparticles-Coated Paclitaxel Nanoliposomes and Their Antitumor Efficacy

To functionalize the advanced controlled drug delivery systems, gold nanomaterials have been explored as photothermal therapy agents owing to their tunable and strong localized surface plasmon resonance (LSPR). As the major tissue chromophores are barely absorptive in the near-infrared (NIR) region (650–900 nm), it is becoming particularly important to regulate the LSPR absorption of gold nanomaterials to the NIR region to minimize the absorption. The main factors for the LSPR absorption of nanomaterials, which decides the photothermal effects, drug release, and antitumor efficacy of gold-based drug carriers, include morphology, size, and dielectric constant of surrounding mediums. Herein, gold nanoparticles-coated paclitaxel nanoliposomes (PTX-Lips@AuNPs) with various morphologies, sizes, and dielectric constant of surrounding mediums were synthesized. We studied the regulatory mechanism of LSPR absorption and their antitumor efficacy. The results showed that the branched gold nanoshells-coated paclitaxel nanoliposomes (PTX-Lips@BGNs) with a big size (about 150 nm) in the medium of NaCl solution exhibit optimal antitumor efficacy among different parameters we have studied. Furthermore, the investigation of the regulatory mechanism endows a synthetic theoretical basis of the nanomaterials in the future.

Discovery screen platform for co-modulators of T cell effector function against tumors

Abstract A major goal for cancer immunotherapy is to target pathways that drive optimal anti-tumor efficacy. T cell checkpoint therapy aims to enhance T cell responses during priming and/or within tumor tissue, but the key drivers of efficacy in broad patient populations has remained elusive. Here we first sought to determine whether responsiveness to anti-PD1 therapy in established syngeneic murine tumors depends on recruitment of T cells from lymphoid organs during treatment, or if PD1 blockade on T cells already within tumors is sufficient to drive efficacy. We compared anti-PD1 efficacy on established MC38 tumors when FTY720 (S1PR antagonist that blocks lymphocyte egress from lymphoid organs) or vehicle control was administered from the start of treatment. We found that anti-PD1 efficacy did not require additional T cells migrating from lymphoid organs, suggesting that PD1 blockade can act directly on T cells that were already present within tumor tissue. We next examined the functionality of tumor-infiltrating T cell subsets during anti-PD1 treatment and found that cytokine production is enhanced in a population of PD1intermediate T cells, but not on PD1high T cells. Together, this suggests that PD1 checkpoint therapy is driven by amelioration of a fraction of PD1+ tumor-infiltrated T cells, and that additional pathways within established tumors may need to be co-targeted to drive optimal efficacy. We then developed an in vitro discovery screening platform to identify pathways that co-modulate human T cell effector function against PDL1+ cells under conditions that drive high levels of PD1 on T cells. We are currently screening genes of interest that are expressed in patient tumor tissues and highly associated with a T cell-inflamed signature.