Small-Molecule Therapeutics Achieve Multiple Mechanism-Mediated Sensitizations of Colorectal Cancer to Immune Checkpoint Blockade via Neutrophil Micropharmacies.

Immune checkpoint blockade (ICB) therapy has been approved for colorectal cancer (CRC). However, response rates are variable and often <50%. The low tumor immunogenicity and immunosuppressive tumor microenvironment (TME) jointly contribute to this suboptimal response rate. This study confirmed the potential of combining immunogenic cell death (ICD) inducer irinotecan (IRI) and transforming growth factor-ß (TGF-ß) inhibitor galunisertib (GAL) to improve tumor immunogenicity and remodel the immunosuppressive TME. Moreover, to ameliorate the in vivo delivery barriers associated with small molecules, neutrophil micropharmacies (NOG) were developed for the codelivery of IRI and GAL, which loaded the commercial liposome formulation of IRI (ONIVYDE, ONI) intracellularly and conjugated the pH-responsive GAL liposome (GLP) on the cell surface. This neutrophil-based formulation resulted in a >4-fold increase in the ratios of the amount of both IRI and GAL accumulated in tumors to the dosage administration, effectively achieving multiple mechanism-mediated sensitization of CRC to ICB therapy.

Spatiotemporally Controlled T-Cell Combination Therapy for Solid Tumor.

Due to multidimensional complexity of solid tumor, development of rational T-cell combinations and corresponding formulations is still challenging. Herein, a triple combination of T cells are developed with Indoleamine 2,3-dioxygenase inhibitors (IDOi) and Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). To maximize synergism, a spatiotemporally controlled T-cell engineering technology to formulate triple drugs into one cell therapeutic, is established. Specifically, a sequentially responsive core-shell nanoparticle (SRN) encapsulating IDOi and CDK4/6i is anchored onto T cells. The yielded SRN-T cells migrated into solid tumor, and achieved a 1st release of IDOi in acidic tumor microenvironment (TME). Released IDOi restored tryptophan supply in TME, which activated effector T cells and inhibited Tregs. Meanwhile, 1st released core is internalized by tumor cells and degraded by glutathione (GSH), to realize a 2nd release of CDK4/6i, which induced up-regulated expression of C-X-C motif chemokine ligand 10 (CXCL10) and C-C motif chemokine ligand 5 (CCL5), and thus significantly increased tumor infiltration of T cells. Together, with an enhanced recruitment and activation, T cells significantly suppressed tumor growth, and prolonged survival of tumor-bearing mice. This study demonstrated rationality and superiority of a tri-drug combination mediated by spatiotemporally controlled cell-engineering technology, which provides a new treatment regimen for solid tumor.

Enhancing Glioblastoma Immunotherapy with Integrated Chimeric Antigen Receptor T Cells through the Re-Education of Tumor-Associated Microglia and Macrophages.

Glioblastoma (GBM) is an aggressive brain cancer that is highly resistant to treatment including chimeric antigen receptor (CAR)-T cells. Tumor-associated microglia and macrophages (TAMs) are major contributors to the immunosuppressive GBM microenvironment, which promotes tumor progression and treatment resistance. Hence, the modulation of TAMs is a promising strategy for improving the immunotherapeutic efficacy of CAR-T cells against GBM. Molecularly targeting drug pexidartinib (PLX) has been reported to re-educate TAMs toward the antitumorigenic M1-like phenotype. Here, we developed a cell-drug integrated technology to reversibly conjugate PLX-containing liposomes (PLX-Lip) to CAR-T cells and establish tumor-responsive integrated CAR-T cells (PLX-Lip/AZO-T cells) as a combination therapy for GBM. We used a mouse model of GBM to show that PLX-Lip was stably maintained on the surface of PLX-Lip/AZO-T cells in circulation and these cells could transmigrate across the blood-brain barrier and deposit PLX-Lip at the tumor site. The uptake of PLX-Lip by TAMs effectively re-educated them into the M1-like phenotype, which in turn boosted the antitumor function of CAR-T cells. GBM tumor growth was completely eradicated in 60% of the mice after receiving PLX-Lip/AZO-T cells and extended their overall survival time beyond 50 days; in comparison, the median survival time of mice in other treatment groups did not exceed 35 days. Overall, we demonstrated the successful fusion of CAR-T cells and small-molecule drugs with the cell-drug integrated technology. These integrated CAR-T cells provided a superior combination strategy for GBM treatment and presented a reference for the construction of integrated cell-based drugs.

Sensitizing Tumors to Immune Checkpoint Blockage via STING Agonists Delivered by Tumor-Penetrating Neutrophil Cytopharmaceuticals.

Immune checkpoint inhibitors (ICIs) have displayed potential efficacy in triple-negative breast cancer (TNBC) treatment, while only a minority of patients benefit from ICI therapy currently. Although activation of the innate immune stimulator of interferon genes (STING) pathway potentiates antitumor immunity and thus sensitizes tumors to ICIs, the efficient tumor penetration of STING agonists remains critically challenging. Herein, we prepare a tumor-penetrating neotype neutrophil cytopharmaceutical (NEs@STING-Mal-NP) with liposomal STING agonists conjugating on the surface of neutrophils, which is different from the typical neutrophil cytopharmaceutical that loads drugs inside the neutrophils. We show NEs@STING-Mal-NP that inherit the merits of neutrophils including proactive tumor vascular extravasation and tissue penetration significantly boost the tumor penetration of STING agonists. Moreover, the backpacked liposomal STING agonists can be released in response to hyaluronidase rich in the tumor environment, leading to enhanced uptake by tumor-infiltrating immune cells and tumor cells. Thus, NEs@STING-Mal-NP effectively activate the STING pathway and reinvigorate the tumor environment through converting macrophages and neutrophils to antitumor phenotypes, promoting the maturation of dendritic cells, and enhancing the infiltration and tumoricidal ability of T cells. Specifically, this cytopharmaceutical displays a significant inhibition on tumor growth and prolongs the survival of TNBC-bearing mice when combined with ICIs. We demonstrate that neutrophils serve as promising vehicles for delivering STING agonists throughout solid tumors and the developed neutrophil cytopharmaceuticals with backpacked STING agonists exhibit huge potential in boosting the immunotherapy of ICIs.

Real-time detection of T cell activation by visualizing TCR nanoclusters with a cholesterol derived aggregation-induced emission probe.

Successful T-cell based immunotherapy usually depends on the activation of T cells. Most of commonly used methods for assessing T cell activity rely on the antibody-based technology, which focus on detecting protein-centered activation markers, including CD25, cytokines and so on. However, these methods always involve tedious sample-preparation process, labor-consuming and costly, which could not be utilized in real-time detection. The T cell receptor (TCR) clustering is another kind of essential T cell activation marker on the membrane, which increases during the activation state of T cells. We herein developed a cholesterol derived aggregation-induced emission (AIE) fluorescent probe (R-TPE-PEG-Chol) for detecting T cell activation in real-time. Five probes were first designed and synthesized and among them COOH-TPE-PEG-Chol displayed the best imaging effects, which had no significant impact on the key physiological functions of T cells. In addition, we have proved that COOH-TPE-PEG-Chol was introduced onto the naïve T cell membrane in its molecularly dissolved form without fluorescent emission. While during T cell activation, the formation of TCR nanoclusters would induce aggregation of membrane cholesterol, which could provoke the fluorescence signal of the COOH-TPE-PEG-Chol due to the AIE characteristic. Moreover, the enhancement of the fluorescence intensity was positively related to the activation state of T cells. Our study demonstrated the concept of cholesterol-derived AIE fluorescent probes for deciphering the spatiotemporal arrangements of TCR on the membrane during T cell activation, and consequently provided a novel and complementary strategy for detecting T cell activation in real-time.

An EPR-Independent extravasation Strategy: Deformable leukocytes as vehicles for improved solid tumor therapy.

Effective delivery of therapeutic modality throughout the tumorous nidus plays a crucial role in successful solid tumor treatment. However, conventional nanomedicines based on enhanced permeability and retention (EPR) effect have yielded limited delivery/therapeutic efficiency, due mainly to the heterogeneity of the solid tumor. Leukocytes, which could intrinsically migrate across the vessel wall and crawl through tissue interstitium in a self-deformable manner, have currently emerged as an alternative drug delivery vehicle. In this review, we start with the intrinsic properties of leukocytes (e.g., extravasation and crawling inside tumor), focusing on unveiling the conceptual rationality of leveraging leukocytes as EPR-independent delivery vehicles. Then we discussed various cargoes-loading/unloading strategies for leukocyte-based vehicles as well as their promising applications. This review aims to serve as an up-to-date compilation, which might provide inspiration for scientists in the field of drug delivery.

Modular synthesis of amphiphilic chitosan derivatives based on copper-free click reaction for drug delivery.

Amphiphilic chitosan derivatives have attracted wide attention as drug carriers due to their physicochemical properties. However, obtaining a desired amphiphilic chitosan derivative by tuning the various functional groups was complex and time-consuming. Therefore, a facile and common synthesis strategy would be promising. In this study, a modular strategy based on strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction was designed and applied in synthesizing deoxycholic acid- or octanoic acid-modified N-azido propionyl-N,O-sulfate chitosan through tuning the hydrophobic groups. Additionally, chitosan derivatives with the same substitute groups were prepared via amide coupling as controls. We demonstrated that these derivates via the two strategies showed no obvious difference in physicochemical properties, drug loading ability and biosafety, indicating the feasibility of modular strategy. Notably, the modular strategy exhibited advantages including high reactivity, flexibility and reproducibility. We believe that this modular strategy could provide varied chitosan derivatives in an easy and high-efficiency way for improving multifunctional drug carriers.

Novel Nonsecosteroidal Vitamin D Receptor Modulator Combined with Gemcitabine Enhances Pancreatic Cancer Therapy through Remodeling of the Tumor Microenvironment.

In a pancreatic tumor microenvironment, activated pancreatic stellate cells (PSCs) produce extracellular matrix (ECM) to form a barrier to drug penetration. Moreover, the interaction between cancer cells and activated PSCs promotes the tumor growth. Vitamin D receptor (VDR), as a key regulator to promote the recovery of PSCs to the resting state, is an attractive therapeutic target for pancreatic cancer. Herein, we reported the design and synthesis of 57 nonsecosteroidal VDR modulators based on the skeleton of phenyl-pyrrolyl pentane. Among them, compounds C4, I5, and I8 exhibited excellent VDR affinity and effective inhibition of the activation of PSCs, as well as potent suppression of the interaction between cancer cells and PSCs in vitro. In vivo, compound I5 combined with gemcitabine achieved efficacious antitumor activity without causing hypercalcemia. In conclusion, the compounds designed in our study can remodel the tumor microenvironment and are expected to be candidates for the treatment of pancreatic cancer.

Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy.

Treatment of solid tumors with T cell therapy has yielded limited therapeutic benefits to date. Although T cell therapy in combination with proinflammatory cytokines or immune checkpoints inhibitors has demonstrated preclinical and clinical successes in a subset of solid tumors, unsatisfactory results and severe toxicities necessitate the development of effective and safe combinatorial strategies. Here, the liposomal avasimibe (a metabolism-modulating drug) was clicked onto the T cell surface by lipid insertion without disturbing the physiological functions of the T cell. Avasimibe could be restrained on the T cell surface during circulation and extravasation and locally released to increase the concentration of cholesterol in the T cell membrane, which induced rapid T cell receptor clustering and sustained T cell activation. Treatment with surface anchor-engineered T cells, including mouse T cell receptor transgenic CD8+ T cells or human chimeric antigen receptor T cells, resulted in superior antitumor efficacy in mouse models of melanoma and glioblastoma. Glioblastoma was completely eradicated in three of the five mice receiving surface anchor-engineered chimeric antigen receptor T cells, whereas mice in other treatment groups survived no more than 64 days. Moreover, the administration of engineered T cells showed no obvious systemic side effects. These cell-surface anchor-engineered T cells hold translational potential because of their simple generation and their safety profile.

Cytopharmaceuticals: An emerging paradigm for drug delivery.

Cytopharmaceuticals, in which drugs/nanomedicines are loaded into/onto autologous patient- or allogeneic donor-derived living cells ex vivo, have displayed great promise for targeted drug delivery in terms of improved biocompatibility, superior targeting, and prolonged circulation. Despite certain impressive therapeutic benefits in preclinical studies, several obstacles retard their clinical application, such as the lack of facile and convenient methods of carrier cell acquisition, technologies for preparing cytopharmaceuticals at scale with undisturbed carrier cell viability, and modalities for monitoring the in vivo fate of cytopharmaceuticals. To comprehensively understand cytopharmaceuticals and thereby accelerate their clinical translation, this review covers the main sources of various cytopharmaceuticals, technologies for preparing cytopharmaceuticals, the in vivo fate of cytopharmaceuticals including carrier cells and loaded drugs/nanomedicines, and the application prospects of cytopharmaceuticals. It is our hope that this review will elucidate the bottlenecks associated with cytopharmaceutical preparation, leading to the acceleration of future industrialization of cell-based formulations.

Sulfonyl-containing phenyl-pyrrolyl pentane analogues: Novel non-secosteroidal vitamin D receptor modulators with favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity.

Modulating the vitamin D receptor (VDR) is an effective way to treat for cancer. We previously reported a potent non-secosteroidal VDR modulator (sw-22) with modest anti-tumor activity, which could be due to its undesirable physicochemical and pharmacokinetic properties. In this study, we investigated the structure-activity and structure-property relationships around the 2'-hydroxyl group of sw-22 to improve the physicochemical properties, pharmacokinetic properties and anti-tumor activity. Compounds 19a and 27b, the potent non-secosteroidal VDR modulators, were identified as the most effective molecules in inhibiting the proliferation of three cancer cell lines, particularly breast cancer cells, with a low IC50 via the distribution of cell cycle and induction of apoptosis by stimulating the expression of p21, p27 and Bax. Further investigation revealed that 19a and 27b possessed favorable rat microsomal metabolic stability (2.22 and 2.3 times, respectively, more stable than sw-22), solubility (43.9 and 50.2 times, respectively, more soluble than sw-22) and in vivo pharmacokinetic properties. In addition, 19a and 27b showed excellent in vivo anti-tumor activity without cause hypercalcemia, which is the main side effect of marketed VDR modulators. In summary, the favorable physicochemical properties, pharmacokinetic properties and anti-tumor activity of 19a and 27b highlight their potential therapeutic applications in cancer treatment.

Further Developments of the Phenyl-Pyrrolyl Pentane Series of Nonsteroidal Vitamin D Receptor Modulators as Anticancer Agents.

The vitamin D3 receptor (VDR), which belongs to the nuclear-receptor superfamily, is a potential molecular target for anticancer-drug discovery. In this study, a series of nonsteroidal vitamin D mimics with phenyl-pyrrolyl pentane skeletons with therapeutic potentials in cancer treatment were synthesized. Among them, 11b and 11g were identified as the most effective agents in reducing the viability of four cancer-cell lines, particularly those of breast-cancer cells, with IC50 values in the submicromolar-concentration range. In addition, 11b and 11g possessed VDR-binding affinities and displayed significant partial VDR-agonistic activities determined by dual-luciferase-reporter assays and human-leukemia-cell-line (HL-60)-differentiation assays. Furthermore, 11b and 11g inhibited tumor growth in an orthotopic breast-tumor model via inhibition of cell proliferation and induction of cell apoptosis. More importantly, 11b and 11g exhibited favorable pharmacokinetic behavior in vivo and did not increase serum calcium levels or cause any other apparent side effects. In summary, 11b and 11g act as novel VDR modulators and may be promising candidates for cancer chemotherapy.

Design, synthesis and biological evaluation of nonsecosteroidal vitamin D3 receptor ligands as anti-tumor agents.

1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3, also known as calcitriol), the active form of vitamin D3, is being increasingly recognized for cancer therapy. Our previous work showed that phenyl-pyrrolyl pentane analogs, which mimicked anti-proliferative activities against several cancer cell lines of the natural secosteroidal ligand 1,25-(OH)2D3. Here, in order to optimize the structural features and discover more potent derivative, a series of nonsecosteroidal vitamin D3 receptor (VDR) ligands bearing acetylene bond linker was designed, synthesized and evaluated. Most of them showed moderate to good binding affinities and agonistic activities. Especially, compound 19f displayed the most anti-proliferative activities against MCF-7 and PC-3 cells with the IC50 values of 1.80 and 5.35µM, respectively, which was comparable to positive control 1,25-(OH)2D3. Moreover, compound 19f exhibited reduced toxicity against human normal liver cell line (L02) compared with the parental compound 7. Besides, the preliminary structure-activity relationships (SARs) were also analyzed.

Design, synthesis and evaluation of pyrrolo[2,3-d]pyrimidine-phenylamide hybrids as potent Janus kinase 2 inhibitors.

Janus kinase 2 (JAK2) plays an essential role in the signaling of hormone-like cytokines and growth factors, which has been convinced as an important target of myeloproliferative neoplasms (MPNs) therapy. In this study, a series of novel pyrrolo[2,3-d]pyrimidine-phenylamide hybrids were designed and synthesized as potential JAK2 inhibitors through hybridization strategy. In vitro biological studies showed that most of these compounds exhibited potent activity against JAK2. Especially, compound 16c was identified as a suitable lead compound, which showed favorable pharmacokinetic profiles in rats (F=73.57%), excellent in vitro efficacy against erythroleukemic cells (TF-1, IC50=0.14µM), and high selectivity for JAK2 (IC50=6nM with >97-fold selectivity vs JAK3).

Novel nonsecosteroidal VDR ligands with phenyl-pyrrolyl pentane skeleton for cancer therapy.

A series of nonsecosteroidal vitamin D3 receptor (VDR) ligands with phenyl-pyrrolyl pentane skeleton were synthesized for cancer therapy. In contrast to 1α,25-dihydroxyvitamin D3 (Calcitriol), these VDR ligands exhibited anti-proliferative activity without inducing hypercalcemia. These compounds were evaluated for vitamin D3-agonistic ability and anti-proliferative activity in vitro. Among them, compounds 5k and 5i exhibited equivalent vitamin D3-agonistic activity compared with Calcitriol. Meanwhile, compound 5k displayed promising inhibiting profile against MCF-7, HepG-2 and Caco-2 with IC50 values of 0.00586 µM, 0.176 µM, and 1.01 µM (Calcitriol: 5.58 µM, 80.83 µM and 4.46 µM) respectively. Compound 5i inhibited proliferation of PC-3 with IC50 value of 0.00798 µM (Calcitriol: 17.25 µM). Additionally, neither of these compounds significantly elevated serum calcium in rats.