A Single-Domain TCR-like Antibody Selective for the Qa-1b/Qdm Peptide Complex Enhances Tumoricidal Activity of NK Cells via Blocking the NKG2A Immune Checkpoint.

The NKG2A/HLA-E axis is an immune checkpoint that suppresses immune effector activity in the tumor microenvironment. In mice, the ligand for the NKG2A/CD94 inhibitory receptor is the nonclassical MHC molecule Qa-1b, the HLA-E ortholog, which presents the peptide AMAPRTLLL, referred to as Qdm (for Qa-1 determinant modifier). This dominant peptide is derived from the leader sequences of murine classical MHC class I encoded by the H-2D and -L loci. To broaden our understanding of Qa-1b/Qdm peptide complex biology and its tumor protective role, we identified a TCR-like Ab from a single domain VHH library using yeast surface display. The TCR-like Ab (EXX-1) binds only to the Qa-1b/Qdm peptide complex and not to Qa-1b alone or Qa-1b loaded with control peptides. Conversely, currently available Abs to Qa-1b bind independent of peptide loaded. Flow cytometric results revealed that EXX-1 selectively bound to Qa-1b/Qdm-positive B16F10, RMA, and TC-1 mouse tumor cells but only after pretreatment with IFN-γ; no binding was observed following genetic knockdown of Qa-1b or Qdm peptide. Furthermore, EXX-1 Ab blockade promoted NK cell-mediated tumor cell lysis in vitro. Our findings show that EXX-1 has exquisite binding specificity for the Qa-1b/Qdm peptide complex, making it a valuable research tool for further investigation of the Qa-1b/Qdm peptide complex expression and regulation in healthy and diseased cells and for evaluation as an immune checkpoint blocking Ab in syngeneic mouse tumor models.

Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor.

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

Detection of specific antibody-ligand interactions with a self-induced back-action actuated nanopore electrophoresis sensor.

Recent advances in plasmonic nanopore technologies have enabled the use of concurrently acquired bimodal optical-electrical data for improved quantification of molecular interactions. This work presents the use of a new plasmonic nanosensor employing self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) for quantifying antibody-ligand interactions. T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting major histocompatibility complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells, were used to test the SANE sensor's ability to identify specific antibody-ligand binding. Cancer-irrelevant TCRmAbs targeting the same pMHCs were also tested as a control. It was found that the sensor could provide bimodal molecular signatures that could differentiate between antibody, ligand and the complexes that they formed, as well as distinguish between specific and non-specific interactions. Furthermore, the results suggested an interesting phenomenon of increased antibody-ligand complex bound fraction detected by the SANE sensor compared to that expected for corresponding bulk solution concentrations. A possible physical mechanism and potential advantages for the sensor's ability to augment complex formation near its active sensing volume at concentrations lower than the free solution equilibrium binding constant (K D ) are discussed.

Targeted chemotherapy via HER2-based chimeric antigen receptor (CAR) engineered T-cell membrane coated polymeric nanoparticles.

Cell membrane-derived nanoparticles (NPs) have recently gained popularity due to their desirable features in drug delivery such as mimicking properties of native cells, impeding systemic clearance, and altering foreign body responses. Besides NP technology, adoptive immunotherapy has emerged due to its promise in cancer specificity and therapeutic efficacy. In this research, we developed a biomimetic drug carrier based on chimeric antigen receptor (CAR) transduced T-cell membranes. For that purpose, anti-HER2 CAR-T cells were engineered via lentiviral transduction of anti-HER2 CAR coding lentiviral plasmids. Anti-HER2 CAR-T cells were characterized by their specific activities against the HER2 antigen and used for cell membrane extraction. Anti-cancer drug Cisplatin-loaded poly (D, l-lactide-co-glycolic acid) (PLGA) NPs were coated with anti-human epidermal growth factor receptor 2 (HER2)-specific CAR engineered T-cell membranes. Anti-HER2 CAR-T-cell membrane-coated PLGA NPs (CAR-T-MNPs) were characterized and confirmed via fluorescent microscopy and flow cytometry. Membrane-coated NPs showed a sustained drug release over the course of 21 days in physiological conditions. Cisplatin-loaded CAR-T-MNPs also inhibited the growth of multiple HER2+ cancer cells in vitro. In addition, in vitro uptake studies revealed that CAR-T-MNPs showed an increased uptake by A549 cells. These results were also confirmed via in vivo biodistribution and therapeutic studies using a subcutaneous lung cancer model in nude mice. CAR-T-MNPs localized preferentially at tumor areas compared to those of other studied groups and consisted of a significant reduction in tumor growth in tumor-bearing mice. In Conclusion, the new CAR modified cell membrane-coated NP drug-delivery platform has demonstrated its efficacy both in vitro and in vivo. Therefore, CAR engineered membrane-coated NP system could be a promising cell-mimicking drug carrier that could improve therapeutic outcomes of lung cancer treatments.

The MHC-E peptide ligands for checkpoint CD94/NKG2A are governed by inflammatory signals, whereas LILRB1/2 receptors are peptide indifferent.

The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.

Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor for Label-Free Detection of Cancer Immunotherapy-Relevant Antibody-Ligand Interactions.

We fabricated a novel single molecule nanosensor by integrating a solid-state nanopore and a double nanohole nanoaperture. The nanosensor employs Self-Induced Back-Action (SIBA) for optical trapping and enables SIBA-Actuated Nanopore Electrophoresis (SANE) for concurrent acquisition of bimodal optical and electrical signatures of molecular interactions. This work describes how to fabricate and use the SANE sensor to quantify antibody-ligand interactions. We describe how to analyze the bimodal optical-electrical data to improve upon the discrimination of antibody and ligand versus bound complex compared to electrical measurements alone. Example results for specific interaction detection are described for T-cell receptor-like antibodies (TCRmAbs) engineered to target peptide-presenting Major Histocompatibility Complex (pMHC) ligands, representing a model of target ligands presented on the surface of cancer cells. We also describe how to analyze the bimodal optical-electrical data to discriminate between specific and non-specific interactions between antibodies and ligands. Example results for non-specific interactions are shown for cancer-irrelevant TCRmAbs targeting the same pMHCs, as a control. These example results demonstrate the utility of the SANE sensor as a potential screening tool for ligand targets in cancer immunotherapy, though we believe that its potential uses are much broader.