Aptamer-Assisted Blockade of the Immune Suppressor Sialic Acid-Binding Immunoglobulin-Like Lectin-15 for Cancer Immunotherapy

The percentage of low response and adaptive resistance to current antibody-based immune checkpoint blockade (ICB) therapy requires the development of novel immunotherapy strategies. Here, we developed an aptamer-assisted immune checkpoint blockade (Ap-ICB) against sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15), a novel immune suppressor broadly upregulated on cancer cells and tumor infiltrating myeloid cells, which is mutually exclusive of programmed cell death ligand 1 (PD-L1). Using protein aptamer selection, we identified WXY3 aptamer with high affinity against Siglec-15 protein/Siglec-15 positive cells. We demonstrated that WXY3 aptamer rescued antigen-specific T cell responses in vitro and in vivo. Importantly, the WXY3 Ap-ICB against Siglec-15 amplified anti-tumor immunity in the tumor microenvironment and inhibited tumor growth/metastasis in syngeneic mouse model, which may result from enhanced macrophage and T cell functionality. In addition, by using aptamer-based spherical nucleic acids, we developed a synergetic ICB strategy of multivalent binding and steric hindrance, which further improves the in vivo anti-tumor effect. Taken together, our results support Ap-ICB targeted Siglec-15 as a potential strategy for normalization cancer immunotherapy.     

A novel peptide-based probe 99mTc-PEG6-RD-PDP2 for the molecular imaging of tumor PD-L2 expression

Tumor-related PD-L2 expression is associated with the clinical efficacy of PD-1/PD-L1 blockade therapy. PD-L2-specific imaging can help selecting patients for appropriate immunotherapy. In this study, a PD-L2-targeting peptide (PDP2) was screened by the one-bead one-compound combinatorial library approach. Using the retro-inverso d-peptide of PDP2 (RD-PDP2) and PEGylation strategies, we developed a novel Tc-99m-labeled PD-L2-targeting peptide as a SPECT tracer (^99mTc-PEG₆-RD-PDP2) for imaging of tumor PD-L2 expression. The radiolabeling yield of ^99mTc-PEG₆-RD-PDP2 was greater than 95% by the standard HYNIC/tricine/TPPTS labeling procedure. ^99mTc-PEG₆-RD-PDP2 displayed high PD-L2-binding specificity both in vitro and in vivo. SPECT/CT imaging with ^99mTc-PEG₆-RD-PDP2 showed that the A549-PD-L2 tumors were clearly visualized, whereas the signals in PD-L2-negative A549 tumors were much lower. In vivo blocking study suggested that the tumor uptake of ^99mTc-PEG₆-RD-PDP2 was PD-L2 specifically mediated. ^99mTc-PEG₆-RD-PDP2 is a promising SPECT probe for the non-invasive imaging of tumor PD-L2 expression and has a great potential in guiding the anti-PD-1 or anti-PD-L1 immunotherapy of cancer.     

Novel Complex of PD-L1 Aptamer and Albumin Enhances Antitumor Efficacy In Vivo

The PD-1/PD-L1 pathway blockade can generate a good clinical response by reducing immunosuppression and provoking durable antitumor immunity. In addition to antibodies, aptamers can also block the interaction between PD-1 and PD-L1. For the in vivo application, however, free aptamers are usually too small in size and quickly removed from blood via glomerular filtration. To avoid renal clearance of aptamer, we conjugated the PD-L1 aptamer to albumin to form a larger complex (BSA-Apt) and evaluated whether BSA-Apt would enhance the in vivo antitumor efficacy. The PD-L1 aptamer was thiol-modified and conjugated to the amino group of BSA via a SMCC linker. The average size of BSA-Apt was 11.65 nm, which was above the threshold for renal clearance. Functionally, BSA-Apt retained the capability of the PD-L1 aptamer to bind with PDL1-expressing tumor cells. Moreover, both the free aptamer and BSA-Apt augmented the PBMC-induced antitumor cytotoxicity in vitro. Furthermore, BSA-Apt generated a significantly stronger antitumor efficacy than the free PD-L1 aptamer in vivo without raising systemic toxicity. The results indicate that conjugating the PD-L1 aptamer to albumin may serve as a promising strategy to improve the in vivo functionality of the aptamer and that BSA-Apt may have application potential in cancer immunotherapy.     

A photoresponsive antibody–siRNA conjugate for activatable immunogene therapy of cancer

Tumor-targeted delivery of small-interfering RNAs (siRNAs) for cancer therapy still remains a challenging task. While antibody–siRNA conjugates (ARCs) provide an alternative way to address this challenge, the uncontrollable siRNA release potentially leads to undesirable off-tumor side effects, limiting their in vivo therapeutic efficacy. Here, we report a photoresponsive ARC (PARC) for tumor-specific and photoinducible siRNA delivery as well as photoactivable immunogene therapy. PARC is composed of an anti-programmed death-ligand 1 antibody (αPD-L1) conjugated with a siRNA against intracellular PD-L1 mRNA through a photocleavable linker. After targeting cancer cells through the interaction between αPD-L1 and membrane PD-L1, PARC is internalized and it liberates siPD-L1 upon light irradiation to break the photocleavable linker. The released siPD-L1 then escapes from the lysosome into the cytoplasm to degrade intracellular PD-L1 mRNA, which combines the blockade of membrane PD-L1 by αPD-L1 to boost immune cell activity. Owing to these features, PARC causes effective cancer suppression both in vitro and in vivo. This study thus provides a useful conditional delivery platform for siRNAs and a novel means for activatable cancer immunogene therapy.

A photoresponsive antibody–siRNA conjugate (PARC) enables tumor-targeted siRNA delivery and photoactivatable gene silencing for cancer immunotherapy.     

Bisarylmaleimides & the Corresponding Indolocarbazoles as Kinase Inhibitors

Cyclin dependent kinases (CDKs) have recently raised considerable attention because of their central role in the regulation of cell cycle progression. A high incidence of genetic mutation of CDK substrates and deregulation of CDK modulators were found in a number of disease states, particularly in cancer. A novel series of unsymmetrical substituted indolocarbazoles were synthesized and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles were found to b…     

Bioorthogonal Reaction-Mediated Tumor-Selective Delivery of CRISPR/Cas9 System for Dual-Targeted Cancer Immunotherapy

CRISPR system-assisted immunotherapy is an attractive option in cancer therapy. However, its efficacy is still less than expected due to the limitations in delivering the CRISPR system to target cancer cells. Here, we report a new CRISPR/Cas9 tumor-targeting delivery strategy based on bioorthogonal reactions for dual-targeted cancer immunotherapy. First, selective in vivo metabolic labeling of cancer and activation of the cGAS-STING pathway was achieved simultaneously through tumor microenvironment (TME)-biodegradable hollow manganese dioxide (H-MnO₂) nano-platform. Subsequently, CRISPR/Cas9 system-loaded liposome was accumulated within the modified tumor tissue through in vivo click chemistry, resulting in the loss of protein tyrosine phosphatase N2 (PTPN2) and further sensitizing tumors to immunotherapy. Overall, our strategy provides a modular platform for precise gene editing in vivo and exhibits potent antitumor response by boosting innate and adaptive antitumor immunity.     

Homogeneous,Low-volume,Efficient, and Sensitive Quantitation of Circulating Exosomal PD-L1 for Cancer Diagnosis and Immunotherapy Response Prediction

Immunotherapy has revolutionized cancer treatment, but its efficacy is severely hindered by the lack of effective predictors. Herein, we developed a homogeneous, low-volume, efficient, and sensitive exosomal programmed death-ligand 1 (PD-L1, a type of transmembrane protein) quantitation method for cancer diagnosis and immunotherapy response prediction (HOLMES-Exo_PD-L1). The method combines a newly evolved aptamer that efficiently binds to PD-L1 with less hindrance by antigen glycosylation than antibody, and homogeneous thermophoresis with a rapid binding kinetic. As a result, HOLMES-Exo_PD-L1 is higher in sensitivity, more rapid in reaction time, and easier to operate than existing enzyme-linked immunosorbent assay (ELISA)-based methods. As a consequence of an outstanding improvement of sensitivity, the level of circulating exosomal PD-L1 detected by HOLMES-Exo_PD-L1 can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to correlate positively with the metastasis of adenocarcinoma. Overall, HOLMES-Exo_PD-L1 brings a fresh approach to exosomal PD-L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.     

Ultra‐pH‐Responsive and Tumor‐Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti‐Cancer Efficacy

RNA interference (RNAi) gene silencing technologies have shown significant potential for treating various diseases, including cancer. However, clinical success in cancer therapy remains elusive, mainly owing to suboptimal in vivo delivery of RNAi therapeutics such as small interference RNA (siRNA) to tumors. Herein, we developed a library of polymers that respond to a narrow pH change (ultra‐pH‐responsive), and demonstrated the utility of these materials in targeted and deep tumor‐penetrating nanoparticle (NP) for in vivo RNAi. The new NP platform is mainly composed of the following key components: i) internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration; ii) polyethylene glycol (PEG) chains to prolong blood circulation; and iii) sharp pH‐responsive hydrophobic polymer to improve endosome escape. Through systematic studies of structure–function relationship, the optimized RNAi NPs (<70 nm) showed efficient gene silencing and significant inhibition of tumor growth with negligible toxicities in vivo.     

Cyclin-Dependent Kinase 4 and 6 Inhibitors in Cell Cycle Dysregulation for Breast Cancer Treatment

In cell development, the cell cycle is crucial, and the cycle progression’s main controllers are endogenous CDK inhibitors, cyclin-dependent kinases (CDKs), and cyclins. In response to the mitogenic signal, cyclin D is produced and retinoblastoma protein (Rb) is phosphorylated due to activated CDK4/CDK6. This causes various proteins required in the cell cycle progression to be generated. In addition, complexes of CDK1-cyclin A/B, CDK2-cyclin E/A, and CDK4/CDK6-cyclin D are required in each phase of this progression. Cell cycle dysregulation has the ability to lead to cancer. Based on its role in the cell cycle, CDK has become a natural target of anticancer therapy. Therefore, understanding the CDK structures and the complex formed with the drug, helps to foster the development of CDK inhibitors. This development starts from non-selective CDK inhibitors to selective CDK4/CDK6 inhibitors, and these have been applied in clinical cancer treatment. However, these inhibitors currently require further development for various hematologic malignancies and solid tumors, based on the results demonstrated. In drug development, the main strategy is primarily to prevent and asphyxiate drug resistance, thus a determination of specific biomarkers is required to increase the therapy’s effectiveness as well as patient selection suitability in order to avoid therapy failure. This review is expected to serve as a reference for early and advanced-stage researchers in designing new molecules or repurposing existing molecules as CDK4/CDK6 inhibitors to treat breast cancer.     

Discovery of Novel Small-Molecule Inhibitors of PD-1/PD-L1 Interaction via Structural Simplification Strategy

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC₅₀ = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (K_D = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.     

Development of Dual and Selective Degraders of Cyclin‐Dependent Kinases 4 and 6

Cyclin‐dependent kinases 4 and 6 (CDK4/6) are key regulators of the cell cycle, and there are FDA‐approved CDK4/6 inhibitors for treating patients with metastatic breast cancer. However, due to conservation of their ATP‐binding sites, development of selective agents has remained elusive. Here, we report imide‐based degrader molecules capable of degrading both CDK4/6, or selectively degrading either CDK4 or CDK6. We were also able to tune the activity of these molecules against Ikaros (IKZF1) and Aiolos (IKZF3), which are well‐established targets of imide‐based degraders. We found that in mantle cell lymphoma cell lines, combined IKZF1/3 degradation with dual CDK4/6 degradation produced enhanced anti‐proliferative effects compared to CDK4/6 inhibition, CDK4/6 degradation, or IKZF1/3 degradation. In summary, we report here the first compounds capable of inducing selective degradation of CDK4 and CDK6 as tools to pharmacologically dissect their distinct biological functions.     

A Novel d-Peptide Identified by Mirror-Image Phage Display Blocks TIGIT/PVR for Cancer Immunotherapy

The low response rate and adaptive resistance of PD-1/PD-L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD-1 in many tumors especially anti-PD-1 resistant tumors. Here, mirror-image phage display bio-panning was performed using the d -enantiomer of TIGIT synthesized by hydrazide-based native chemical ligation. d -peptide ^DTBP-3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. ^DTBP-3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8⁺ T cell dependent manner. More importantly, ^DTBP-3 could inhibit tumor growth and metastasis in anti-PD-1 resistant tumor model. This is the first d -peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.     

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC₅₀ value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.     

A proteome-wide CDK/CRK-specific kinase inhibitor promotes tumor cell death in the absence of cell cycle progression

The identification of molecular determinants of tumor cell survival is an important objective in cancer research. Here, we describe a small-molecule kinase inhibitor (RGB-286147), which, besides inhibiting tumor cell cycle progression, exhibits potent cytotoxic activity toward noncycling tumor cells, but not nontransformed quiescent fibroblasts. Extensive yeast three-hybrid (Y3H)-based proteome/kinome scanning with chemical dimerizers revealed CDK1/2/3/5/7/9 and the less well-characterized CDK-related kinases (CRKs) p42/CCRK, PCTK1/3, and PFTK1 as its predominant targets. Thus, RGB-286147 is a proteome-wide CDK/CRK-specific kinase inhibitor whose further study could yield new insight into molecular determinants of tumor cell survival. Our results also suggest that the [1, 3, 6]-tri-substituted-pyrazolo[3,4-d]-pyrimidine-4-one kinase inhibitor scaffold is a promising template for the rational design of kinase inhibitors with potential applications to disease indications other than cancer, such as neurodegeneration, cardiac hypertrophic growth, and AIDS.     

Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.     

Ratiometrically Designed Nanocarrier to Impact Major Cancer Pathways for Effective Pancreatic Cancer Treatment

To achieve secured in vivo drug synergy in stroma-rich pancreatic cancer(PDAC) is not an easy task. Ji and Liu et al. demonstrate the rational development of a ratiometrically designed nanocarrier to co-package a CDK4/6 cell cycle inhibitor palbociclib(PAL) plus an autophagy inhibitor hydroxychloroquine(HCQ) for efficient PDAC treatment. The lead formulation was obtained using a computer software aided in vitro screening, followed by a remote drug co-import into a lipid-coated mesoporous silica nanoparticle. The ratiometric nanocarrier led to the synchronized pharmacokinetic(PK) profile and effective intratumoral buildup of the drug pair post intravenous injection, which otherwise exhibited distinctly different biodistribution characteristics. The treatment using PAL/HCQ co-delivery nanoparticles yielded the most effective shrinkage of PDAC in subcutaneous and orthotopic PANC1 mouse models. The mechanistic investigation also demonstrated the activation of anti-apoptosis pathway after repetitive nanoparticle injection in mice, which promoted the authors to introduce a small molecule anti-apoptosis inhibitor to further improve the performance of their already potent nanoparticle in the PDAC mouse model. This work has been published online in Nature Communications on August 25, 2020.