Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation.

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

Deciphering single-cell protein secretion and gene expressions by constructing cell-antibody conjugates.

Secreted proteins play critical roles in regulating immune responses, exerting cytotoxic effects on tumor cells, promoting inflammatory processes, and influencing cellular metabolism. Deciphering the intricate relationship between the heterogeneity of secreted proteins and their transcriptional states is pivotal in the study of cellular heterogeneity. Here we proposed a cell-antibody conjugate-based sequencing methodology (Cellab-seq) for joint characterization of secreted proteins and transcriptome. Cellab-seq utilizes a chemoenzymatic strategy to construct cell-antibody conjugates, which enables the capture of secreted proteins and their signal transduction with the incorporation of barcode detection antibodies. We applied Cellab-seq to investigate how gene expression influences the activity of secreted proteins in NK cells. Altogether, this strategy facilitates a nuanced understanding of cellular dynamics under diverse physiological conditions, ultimately contributing to the prevention, diagnosis and treatment of diseases.

Patient-derived Immunocompetent Tumor Organoids: A Platform for Chemotherapy Evaluation in the Context of T-cell Recognition.

Most of the anticancer compounds synthesized by chemists are primarily evaluated for their direct cytotoxic effects at the cellular level, often overlooking the critical role of the immune system. In this study, we developed a patient-derived, T-cell-retaining tumor organoid model that allows us to evaluate the anticancer efficacy of chemical drugs under the synergistic paradigm of antigen-specific T-cell-dependent killing, which may reveal the missed drug hits in the simple cytotoxic assay. We evaluated clinically approved platinum (Pt) drugs and a custom library of twenty-eight PtIV compounds. We observed low direct cytotoxicity of Pt drugs, but variable synergistic effects in combination with immune checkpoint inhibitors (ICIs). In contrast, the majority of PtIV compounds exhibited potent tumor-killing capabilities. Interestingly, several PtIV compounds went beyond direct tumor killing and showed significant immunosynergistic effects with ICIs, outstanding at sub-micromolar concentrations. Among these, Pt-19, PtIV compounds with cinnamate axial ligands, emerged as the most therapeutically potent, demonstrating pronounced immunosynergistic effects by promoting the release of cytotoxic cytokines, activating immune-related pathways and enhancing T cell receptor (TCR) clonal expansion. Overall, this initiative marks the first use of patient-derived immunocompetent tumor organoids to explore and study chemotherapy, advancing their path toward more effective small molecule drug discovery.

Chemoenzymatic Measurement of LacNAc in Single-Cell Multiomics Reveals It as a Cell-Surface Indicator of Glycolytic Activity of CD8+ T Cells.

Despite the rich information about the physiological state of a cell encoded in the dynamic changes of cell-surface glycans, chemical methods to capture specific glycan epitopes at the single-cell level are quite limited. Here, we report a chemoenzymatic method for the single-cell detection of N-acetyllactosamine (LacNAc) by labeling LacNAc with a specific DNA barcode. The chemoenzymatic labeling does not alter the transcriptional status of immune cells and is compatible with multiple scRNA-seq platforms. Integrated analysis of LacNAc and the transcriptome of T cells at the single-cell level reveals that the amount of cell-surface LacNAc is significantly upregulated in activated CD8+ T cells but maintained at basal levels in resting CD8+ T cells (i.e., naive and central memory T cells). Further analysis confirms that LacNAc levels are positively correlated with the glycolytic activity of CD8+ T cells during differentiation. Taken together, our study demonstrates the feasibility of the chemoenzymatic detection of cell-surface glycan in single-cell RNA sequencing-based multiomics with TCR sequence and cell-surface epitope information (i.e., scTCR and CITE-seq), and provides a new way to characterize the biological role of glycan in diverse physiological states.

Chemoenzymatic Tagging of Tn/TF/STF Antigens in Living Systems.

Truncated mucin-type O-glycans, such as Tn-associated antigens, are aberrantly expressed biomarkers of cancer, but remain challenging to target. Reactive antibodies to these antigens either lack high-affinity or are prone to antigen escape. Here, we have developed a robust chemoenzymatic strategy for the global labeling of Tn-associated antigens, i.e. Tn (GalNAcα-O-Ser/Thr), Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, TF) and STF (Neu5Acα2-3Galß1-3GalNAcα-O-Ser/Thr, STF) antigens, in human whole blood with high efficiency and selectivity. This method relies on the use of the O-glycan sialyltransferase ST6GalNAc1 to transfer a sialic acid-functionalized adaptor to the GalNAc residue of these antigens. By tagging, the adaptor functionalized antigens can be easily targeted by customized strategies such as, but not limited to, chimeric antigen receptor T-Cells (CAR-T). We expect this tagging system to find broad applications in cancer diagnostics and targeting in combination with established strategies.

Click-Chemistry-Enabled Nanopipettes for the Capture and Dynamic Analysis of a Single Mitochondrion inside One Living Cell.

The in-depth study of single cells requires the dynamically molecular information in one particular nanometer-sized organelle in a living cell, which is difficult to achieve using current methods. Due to high efficiency of click chemistry, a new nanoelectrode-based pipette architecture with dibenzocyclooctyne at the tip is designed to realize fast conjugation with azide group-containing triphenylphosphine, which targets mitochondrial membranes. The covalent binding of one mitochondrion at the tip of the nanopipette allows a small region of the membrane to be isolated on the Pt surface inside the nanopipette. Therefore, the release of reactive oxygen species (ROS) from the mitochondrion is monitored, which is not interfered by the species present in the cytosol. The dynamic tracking of ROS release from one mitochondrion reveals the distinctive "ROS-induced ROS release" within the mitochondria. Further study of RSL3-induced ferroptosis using nanopipettes provides direct evidence for supporting the noninvolvement of glutathione peroxidase 4 in the mitochondria during RSL3-induced ROS generation, which has not previously been observed at the single-mitochondrion level. Eventually, this established strategy should overcome the existing challenge of the dynamic measurement of one special organelle in the complicated intracellular environment, which opens a new direction for electroanalysis in subcellular analysis.

Ru(bpy)3 2+ -Enabled Cell-Surface Photocatalytic Proximity Labeling toward More Efficient Capture of Physically Interacting Cells.

Intercellular proximity labeling has emerged as a promising approach to enable the study of cell-cell interactions (CCIs), but the efficiency of current platforms is limited. Here, we use Ru(bpy)3 2+ to construct an efficient photocatalytic proximity labeling (PPL) system on the cell surface that allows the highly discriminative CCI detection with spatiotemporal resolution. Through the mechanism study and quantitative characterization on living cells, we demonstrate that the singlet-oxygen (1 O2 ) mechanism is more efficient and specific than the single electron transfer (SET) mechanism in Ru-mediated PPL. Ru(bpy)3 2+ catalysts with different cell-anchoring moieties are prepared to facilitate the catalyst loading on primary cells. Finally, based on this system, we develop a "live" T cell receptor (TCR) multimer with TCR-T cells that could sensitively identify and discriminate cells presenting antigens of different affinity, providing a powerful tool to better understand the heterogeneity of antigen presenting cells.

Trimming Crystallizable Fragment (Fc) Glycans Enables the Direct Enzymatic Transfer of Biomacromolecules to Antibodies as Therapeutics.

Glycoengineering has provided powerful tools to construct site-specific antibody conjugates. However, only small-molecule payloads can be directly transferred to native or engineered antibodies using existing glycoengineering strategies. Herein, we demonstrate that reducing the complexity of crystallizable fragment (Fc) glycans could dramatically boost the chemoenzymatic modification of immunoglobulin G (IgG) via an engineered fucosyltransferase. In this platform, antibodies with Fc glycans engineered to a simple N-acetyllactosamine (LacNAc) disaccharide are successfully conjugated to biomacromolecules, such as oligonucleotides and nanobodies, in a single step within hours. Accordingly, we synthesized an antibody-conjugate-based anti-human epidermal growth factor receptor 2 (HER2)/ cluster of differentiation 3 (CD3) bispecific antibody and used it to selectively destroy patient-derived cancer organoids by reactivating endogenous T lymphocyte cells (T cells) inside the organoid. Our results highlight that this platform is a general approach to construct antibody-biomacromolecule conjugates with translational values.

Antigen-Specific T Cell Detection via Photocatalytic Proximity Cell Labeling (PhoXCELL).

Quantitative detection and characterization of antigen-specific T cells are crucial to our understanding of immune responses as well as the development of new immunotherapies. Herein, we report a spatiotemporally resolved method for the detection and quantification of cell-cell interactions via Photocatalytic proXimity CELl Labeling (PhoXCELL). The biocompatible photosensitizer dibromofluorescein (DBF) was leveraged and optimized as a nongenetic alternative of enzymatic approaches for efficient generation of singlet oxygen upon photoirradiation (520 nm) on the cell surface, which allowed the subsequent labeling of nearby oxidized proteins with primary aliphatic amine-based probes. We demonstrated that DBF-functionalized dendritic cells (DCs) could spatiotemporally label interacting T cells in immune synapses via rapid photoirradiation with quantitatively discriminated interaction strength, which revealed distinct gene signatures for T cells that strongly interact with antigen-pulsed DCs. Furthermore, we employed PhoXCELL to simultaneously detect tumor antigen-specific CD8+ as well as CD4+ T cells from tumor-infiltrating lymphocytes and draining lymph nodes in murine tumor models, enabling PhoXCELL as a powerful platform to identify antigen-specific T cells in T cell receptor (TCR)-relevant personal immunotherapy.

Combining cisplatin and a STING agonist into one molecule for metalloimmunotherapy of cancer.

Mounting evidence suggests that strategies combining DNA-damaging agents and stimulator of interferon genes (STING) agonists are promising cancer therapeutic regimens because they can amplify STING activation and remodel the immunosuppressive tumor microenvironment. However, a single molecular entity comprising both agents has not yet been developed. Herein, we designed two PtIV-MSA-2 conjugates (I and II) containing the DNA-damaging chemotherapeutic drug cisplatin and the innate immune-activating STING agonist MSA-2; these conjugates showed great potential as multispecific small-molecule drugs against pancreatic cancer. Mechanistic studies revealed that conjugate I upregulated the expression of transcripts associated with innate immunity and metabolism in cancer cells, significantly differing from cisplatin and MSA-2. An analysis of the tumor microenvironment demonstrated that conjugate I could enhance the infiltration of natural killer (NK) cells into tumors and promote the activation of T cells, NK cells and dendritic cells in tumor tissues. These findings indicated that conjugate I, which was created by incorporating a Pt chemotherapeutic drug and STING agonist into one molecule, is a promising and potent anticancer drug candidate, opening new avenues for small-molecule-based cancer metalloimmunotherapy.

Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement.

Despite the important breakthroughs of immune checkpoint inhibitors in recent years, the objective response rates remain limited. Here, we synthesize programmed cell death protein-1 (PD-1) antibody-iRGD cyclic peptide conjugate (αPD-1-(iRGD)2) through glycoengineering methods. In addition to enhancing tissue penetration, αPD-1-(iRGD)2 simultaneously engages tumor cells and PD-1+ T cells via dual targeting, thus mediating tumor-specific T cell activation and proliferation with mild effects on non-specific T cells. In multiple syngeneic mouse models, αPD-1-(iRGD)2 effectively reduces tumor growth with satisfactory biosafety. Moreover, results of flow cytometry and single-cell RNA-seq reveal that αPD-1-(iRGD)2 remodels the tumor microenvironment and expands a population of "better effector" CD8+ tumor infiltrating T cells expressing stem- and memory-associated genes, including Tcf7, Il7r, Lef1, and Bach2. Conclusively, αPD-1-(iRGD)2 is a promising antibody conjugate therapeutic beyond antibody-drug conjugate for cancer immunotherapy.

Improved ClickTags enable live-cell barcoding for highly multiplexed single cell sequencing.

Click chemistry-enabled DNA barcoding of cells provides a universal strategy for sample multiplexing in single-cell RNA-seq (scRNA-seq). However, current ClickTags are limited to fixed samples as they only label cells efficiently in methanol. Herein, we report the development of a new protocol for barcoding live cells with improved ClickTags. The optimized reactions barcoded live cells without perturbing their physiological states, which allowed sample multiplexing of live cells in scRNA-seq. The general applicability of this protocol is demonstrated in diversified types of samples, including murine and human primary samples. Up to 16 samples across these two species are successfully multiplexed and demultiplexed with high consistency. The wide applications of this method could help to increase throughput, reduce cost and remove the batch effect in scRNA-seq, which is especially valuable for studying clinical samples from a large cohort.

Use of intercellular proximity labeling to quantify and decipher cell-cell interactions directed by diversified molecular pairs.

FucoID is an intercellular proximity labeling technique for studying cell-cell interactions (CCIs) via fucosyltransferase (FT)-meditated fucosyl-biotinylation, which has been applied to probe antigen-specific dendritic cell (DC)-T cell interactions. In this system, bait cells of interest with cell surface-anchored FT are used to capture the interacting prey cells by transferring a biotin-modified substrate to prey cells. Here, we leveraged FucoID to study CCIs directed by different molecular pairs, e.g., programmed cell death protein-1(PD-1)/programmed cell death protein-ligand-1 (PD-L1), and identify unknown or little studied CCIs, e.g., the interaction of DCs and B cells. To expand the application of FucoID to complex systems, we also synthesized site-specific antibody-based FT conjugate, which substantially improves the ability of FucoID to probe molecular signatures of specific CCI when cells of interest (bait cells) cannot be purified, e.g., in clinical samples. Collectively, these studies demonstrate the general applicability of FucoID to study unknown CCIs in complex systems at a molecular resolution.