Low Picomolar Concentrations Research Articles

A Tag‐Free Platform for Synthesis and Screening of Cyclic Peptide Libraries

AbstractIn the realm of high‐throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post‐screening. Our innovation introduces a tag‐free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity‐enhancing derivatization method, utilized in tandem with nano LC‐MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

A Tag-Free Platform for Synthesis and Screening of Cyclic Peptide Libraries.

In the realm of high-throughput screening (HTS), macrocyclic peptide libraries traditionally necessitate decoding tags, essential for both library synthesis and identifying hit peptide sequences post-screening. Our innovation introduces a tag-free technology platform for synthesizing cyclic peptide libraries in solution and facilitates screening against biological targets to identify peptide binders through unconventional intramolecular CyClick and DeClick chemistries (CCDC) discovered through our research. This combination allows for the synthesis of diverse cyclic peptide libraries, the incorporation of various amino acids, and facile linearization and decoding of cyclic peptide binder sequences. Our sensitivity-enhancing derivatization method, utilized in tandem with nano LC-MS/MS, enables the sequencing of peptides even at exceedingly low picomolar concentrations. Employing our technology platform, we have successfully unearthed novel cyclic peptide binders against a monoclonal antibody and the first cyclic peptide binder of HIV capsid protein responsible for viral infections as validated by microscale thermal shift assays (TSA), biolayer interferometry (BLI) and functional assays.

Customizably designed multibodies neutralize SARS-CoV-2 in a variant-insensitive manner.

The COVID-19 pandemic evolves constantly, requiring adaptable solutions to combat emerging SARS-CoV-2 variants. To address this, we created a pentameric scaffold based on a mammalian protein, which can be customized with up to 10 protein binding modules. This molecular scaffold spans roughly 20 nm and can simultaneously neutralize SARS-CoV-2 Spike proteins from one or multiple viral particles. Using only two different modules targeting the Spike's RBD domain, this construct outcompetes human antibodies from vaccinated individuals' serum and blocks in vitro cell attachment and pseudotyped virus entry. Additionally, the multibodies inhibit viral replication at low picomolar concentrations, regardless of the variant. This customizable multibody can be easily produced in procaryote systems, providing a new avenue for therapeutic development and detection devices, and contributing to preparedness against rapidly evolving pathogens.

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Multifunctional NK Cell-Engaging Antibodies Targeting EGFR and NKp30 Elicit Efficient Tumor Cell Killing and Proinflammatory Cytokine Release.

In this work, we have generated novel Fc-comprising NK cell engagers (NKCEs) that bridge human NKp30 on NK cells to human epidermal growth factor receptor (EGFR) on tumor cells. Camelid-derived VHH single-domain Abs specific for human NKp30 and a humanized Fab derived from the EGFR-specific therapeutic Ab cetuximab were used as binding arms. By combining camelid immunization with yeast surface display, we were able to isolate a diverse panel of NKp30-specific VHHs against different epitopes on NKp30. Intriguingly, NKCEs built with VHHs that compete for binding to NKp30 with B7-H6, the natural ligand of NKp30, were significantly more potent in eliciting tumor cell lysis of EGFR-positive tumor cells than NKCEs harboring VHHs that target different epitopes on NKp30 from B7-H6. We demonstrate that the NKCEs can be further improved with respect to killing capabilities by concomitant engagement of FcγRIIIa and that soluble B7-H6 does not impede cytolytic capacities of all scrutinized NKCEs at significantly higher B7-H6 concentrations than observed in cancer patients. Moreover, we show that physiological processes requiring interactions between membrane-bound B7-H6 and NKp30 on NK cells are unaffected by noncompeting NKCEs still eliciting tumor cell killing at low picomolar concentrations. Ultimately, the NKCEs generated in this study were significantly more potent in eliciting NK cell-mediated tumor cell lysis than cetuximab and elicited a robust release of proinflammatory cytokines, both features which might be beneficial for antitumor therapy.

Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase.

The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular “mix-and-read” enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.

P482: NOVEL BISPECIFIC INNATE CELL ENGAGER AFM28 FOR THE TREATMENT OF CD123 POSITIVE ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME

Background: Novel treatments for patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (HR-MDS) with relapsed or refractory (R/R), or measurable residual disease are urgently needed. Targeting of the surface antigen CD123 expressed on both leukemic blasts and stem cells (LSCs) of most patients allows for depletion of both compartments, which is considered a prerequisite for deep anti-tumor responses and induction of prolonged remission. AFM28 is a novel bispecific Innate Cell Engager (ICE®) designed for bivalent high-affinity binding of CD16A on natural killer (NK) cells, redirecting effector cell cytotoxicity to CD123+ tumor cells. Allogeneic NK cell therapy is emerging as a next-generation treatment with demonstrated activity in R/R AML and a very benign safety profile. Retargeting of NK cells with AFM28 may be a particularly effective treatment strategy in patients with CD123+ AML and HR-MDS. Aims: 1. Preclinical characterization of AFM28 pharmacology and toxicology in vitro and in vivo to support clinical development. 2. Assessment of binding and activation of NK cells by AFM28 to test suitability for combination development with allogeneic NK cell therapy. Methods: Flow cytometry (FC) was used to assess binding and retention of AFM28 on NK cells, NK cell activation (degranulation, IFNγ expression) and depletion of leukemic cells from AML and MDS patient specimens. Quantification of secreted cytokines in leukocyte cultures employed bead-based luminex methodology. In vivo efficacy data were acquired by BLI measurement from a murine AML model. Cynomolgus data were acquired from non-GLP dose range finding and a GLP toxicology study using standard methods (ELISA, IHC, FC). Results: AFM28 induced NK cell activation at low picomolar concentrations and mediated efficacious depletion of CD123+ tumor cell lines and primary leukemic cells, including cells with low CD123 expression. NK cell activation was induced more potently than by an effector-enhanced IgG1 and was accompanied by more pronounced degranulation and IFNγ production. Similarly, AFM28 induced depletion of leukemic cells in patient bone marrow samples, without lysis of CD34+/CD123− cells, suggesting sparing of healthy hematopoietic progenitors. Potent anti-tumor activity was confirmed in vivo in a murine tumor model. In preclinical toxicology models, AFM28-induced target cell depletion was associated with minimal release of inflammatory cytokines, including IL-6, TNFα and IFNγ from leukocytes in vitro, and only low-level release of IL-6 in cynomolgus monkeys, despite efficacious depletion of CD123+ basophils and plasmacytoid dendritic cells (pDCs). Characterization of NK cell binding revealed high-avidity interaction, long cell surface retention, and potent induction of NK cell-mediated tumor cell lysis with no impact on NK cell viability. Summary/Conclusion: These data demonstrate that AFM28 exhibits potent pharmacological activity in vitro and in vivo and efficaciously induces NK cell-mediated depletion of primary leukemic cells. Further, toxicology data suggest good tolerability with a low risk of cytokine release syndrome. Together, these findings support the clinical investigation of AFM28 as a potential novel treatment of CD123+ AML and HR-MDS. A first-in-human trial investigating safety and activity of single agent AFM28 in patients with R/R AML is currently in preparation. In addition, these data support the combination of AFM28 with allogeneic NK cell therapy, which holds potential as an effective, novel treatment strategy.

Transiently gene-modulated cell reporter for ultrasensitive detection of estrogen-like compounds in tap water

Abnormal elevation of indispensable steroid hormone estrogens and exposure to exogenous estrogen-like compounds pose adverse health effects to aquatic animals and human alike. These compounds generally display functionally important estrogenic activity even at extremely low picomolar concentrations. In this study we identified one critical but lethal gene (TAF1) that remarkably represses estrogenic activity. This gene is selected as a candidate for genetically modulating an estrogen-responding cell line. To overcome its lethality, instead of adopting a gene knockout strategy, we developed a transient TAF1 depletion strategy using a designed small interfering RNA. By the transient knockdown of TAF1 in the estrogen-responding reporter cell line, the maximum induction signals for endogenous estrogen 17β-estradiol (E2) and environmental estrogens 17α-ethynyl estradiol (EE2) and bisphenol compounds were enhanced by 4.8–13.3 folds. The limit of detection for EE2 is about 8 × 10−15 mol/L. Moreover, by the established method, trace estrogenic activity (14.7–24.2 pg E2 equivalents (E2Eq)/L) can be detected in a portion of Tap water samples.

Phenyl‐Glutarimides: Alternative Cereblon Binders for the Design of PROTACs

AbstractTargeting cereblon (CRBN) is currently one of the most frequently reported proteolysis‐targeting chimera (PROTAC) approaches, owing to favorable drug‐like properties of CRBN ligands, immunomodulatory imide drugs (IMiDs). However, IMiDs are known to be inherently unstable, readily undergoing hydrolysis in body fluids. Here we show that IMiDs and IMiD‐based PROTACs rapidly hydrolyze in commonly utilized cell media, which significantly affects their cell efficacy. We designed novel CRBN binders, phenyl glutarimide (PG) analogues, and showed that they retained affinity for CRBN with high ligand efficiency (LE >0.48) and displayed improved chemical stability. Our efforts led to the discovery of PG PROTAC 4 c (SJ995973), a uniquely potent degrader of bromodomain and extra‐terminal (BET) proteins that inhibited the viability of human acute myeloid leukemia MV4‐11 cells at low picomolar concentrations (IC50=3 pM; BRD4 DC50=0.87 nM). These findings strongly support the utility of PG derivatives in the design of CRBN‐directed PROTACs.

The chemotherapeutic CX-5461 primarily targets TOP2B and exhibits selective activity in high-risk neuroblastoma

Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity—often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.

Cytotoxic effects and tolerability of gemcitabine and axitinib in a xenograft model for c-myc amplified medulloblastoma

Medulloblastoma is the most common high-grade brain tumor in childhood. Medulloblastomas with c-myc amplification, classified as group 3, are the most aggressive among the four disease subtypes resulting in a 5-year overall survival of just above 50%. Despite current intensive therapy regimens, patients suffering from group 3 medulloblastoma urgently require new therapeutic options. Using a recently established c-myc amplified human medulloblastoma cell line, we performed an in-vitro-drug screen with single and combinatorial drugs that are either already clinically approved or agents in the advanced stage of clinical development. Candidate drugs were identified in vitro and then evaluated in vivo. Tumor growth was closely monitored by BLI. Vessel development was assessed by 3D light-sheet-fluorescence-microscopy. We identified the combination of gemcitabine and axitinib to be highly cytotoxic, requiring only low picomolar concentrations when used in combination. In the orthotopic model, gemcitabine and axitinib showed efficacy in terms of tumor control and survival. In both models, gemcitabine and axitinib were better tolerated than the standard regimen comprising of cisplatin and etoposide phosphate. 3D light-sheet-fluorescence-microscopy of intact tumors revealed thinning and rarefication of tumor vessels, providing one explanation for reduced tumor growth. Thus, the combination of the two drugs gemcitabine and axitinib has favorable effects on preventing tumor progression in an orthotopic group 3 medulloblastoma xenograft model while exhibiting a favorable toxicity profile. The combination merits further exploration as a new approach to treat high-risk group 3 medulloblastoma.

CD123 Expression Patterns and Potential of IMGN632, a CD123-Targeted Antibody Drug Conjugate, in Acute Lymphoblastic Leukemia

Background: The interleukin (IL)-3 receptor α-chain, CD123, is the primary low-affinity subunit of the IL-3 receptor. CD123 is expressed at various levels in hematologic malignancies and has thus become an attractive target of therapy. While CD123 expression in precursor B-lymphoblastic leukemia/lymphoma (B-ALL) has been reported, only limited data in T-ALL exists. Furthermore, the potential of CD123-targeted therapies in ALL remains largely unexplored. In this study, we examine CD123 expression levels in a large cohort of ALL patients and assess the impact of IMGN632, a conjugate of a CD123-binding antibody with the novel DNA-alkylating IGN payload DGN549, on in vitro viability of patient ALL cells and cell lines.Methods: The study cohort (n=188) included 162 B-ALL and 26 T-ALL patients diagnosed and treated at the University of Texas MD Anderson Cancer Center between 12/2012 and 1/2017. Among B-ALL patients, 59 (36%) were Philadelphia chromosome-positive (Ph+). T-ALL patients were further subtyped into the following immunophenotypic categories: early, early T precursor (ETP), thymic, and mature. CD123 expression was evaluated using multiparameter flow cytometry (MFC). Blasts were defined by lineage markers as well as CD45dim blast gate, and CD123 expression was measured for the percentage of positivity and mean fluorescence intensity (MFI) ratio over controls. An arbitrary cutoff of 20% was used for expressing nominal MFC results. Median follow up duration was 21.1 months. CD123 expression was correlated with clinical, laboratory, and outcomes results. The number of binding sites for IMGN632 on ALL blast and cell lines was quantified by QuantiBRITE method. Cytotoxicity of IMGN632 was evaluated after continuous exposure up to five days by WST-8 assays on cell lines or by MFC on primary ALL patient samples.Results: Patients had a median age of 46 years (range, 16-88 years) and included 117 (62%) men. The median CD123 positivity on ALL blasts was 78% (range, 0.3-100) with a median MFI ratio of 11 (range, 0-203). For categorical analysis purposes, we used a standard cutoff of 20% to define positive CD123 expression. Prevalence of CD123 expression was overall higher in B-ALL compared to T-ALL [145 (90%) vs. 11 (42%); p<0.001]. Expression prevalence was highest in Ph+ B-ALL (57/59; 97%) followed by Ph- B-ALL (88/103; 85%) and T-ALL (11/26; 42%). In Ph- B-ALL, CD123 expression correlated with CD34 expression (p=0.002) and lower bone marrow blast count (p=0.003). In T-ALL, CD123 expression correlated with immunophenotypic subtypes (early) (p=0.006). To test the potential of targeting CD123 in ALL, B-ALL cell lines (n=12) which express CD123 at levels similar to AML patient samples were exposed to titrated IMGN632 levels. IMGN632 IC50 values ranged between 0.6 and 20 pM, and the conjugate cytotoxicity was highly CD123-specific, as masking CD123 with a competing anti-CD123 antibody reduced the potency more than 1000-fold. To test the anti-leukemia activity of IMGN632 on primary B-ALL cells, bone marrow cells from B-ALL patients were exposed to IMGN632 following which blasts and non-malignant cells were enumerated by MFC. Notably, IMGN632 eliminated more than 90% of B-ALL blasts in 6 out of 8 samples from newly-diagnosed as well as relapsed-refractory patients at low pico-molar concentrations. Normal cells were not affected by IMGN632 at 100-fold higher concentrations.Conclusions: CD123 expression is prevalent across ALL subtypes, including 90% of B-ALL and 42% of T-ALL. CD123 expression was associated with CD34 expression in B-ALL and immunophenotype in T-ALL. The CD123 targeted antibody-drug conjugate IMGN632 demonstrates promising activity and safety in pre-clinical models of B-ALL._ [Display omitted] DisclosuresDaver:Incyte Corporation: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Immunogen: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy. Zweidler-McKay:ImmunoGen: Employment. Kantarjian:ARIAD: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Khoury:Stemline Therapeutics: Research Funding; Kiromics: Research Funding; Pfizer: Research Funding; Angle: Research Funding.

Discovery of SK-575 as a Highly Potent and Efficacious Proteolysis-Targeting Chimera Degrader of PARP1 for Treating Cancers.

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP1) has a well-established role in the signaling and repair of DNA and is a validated therapeutic target for cancers and other human diseases. Here, we have designed, synthesized, and evaluated a series of small-molecule PARP1 degraders based on the proteolysis-targeting chimera (PROTAC) concept. Our efforts have led to the discovery of highly potent PARP1 degraders, as exemplified by compound 18 (SK-575). SK-575 potently inhibits the growth of cancer cells bearing BRCA1/2 mutations and induces potent and specific degradation of PARP1 in various human cancer cells even at low picomolar concentrations. SK-575 achieves durable tumor growth inhibition in mice when used as a single agent or in combination with cytotoxic agents, such as temozolomide and cisplatin. These data demonstrate that SK-575 is a highly potent and efficacious PARP1 degrader.

An engineered chimeric toxin that cleaves activated mutant and wild-type RAS inhibits tumor growth

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.

Adaptive Flexible Sialylated Nanogels as Highly Potent Influenza A Virus Inhibitors.

Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand–receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK‐II cells.

Single-Measurement Multiplexed Quantification of MicroRNAs from Human Tissue Using Catalytic Hairpin Assembly and Förster Resonance Energy Transfer.

Absolute quantification of microRNAs (miRNAs) or other nucleic acid biomarkers is an important requirement for molecular and clinical biosensing. Emerging technologies with beneficial features concerning simplicity and multiplexing present an attractive route for advancing diagnostic tools toward rapid and low-cost bioanalysis. However, the actual translation into the clinic by miRNA quantification in human samples is often missing. Here, we show that implementing time-gated Förster resonance energy transfer (TG-FRET) into a catalytic hairpin assembly (CHA) can be used for the simultaneous quantification of two miRNAs with a single measurement from total RNA extracts of human tissues. A single terbium-dye FRET pair was conjugated at two specific distances within target-specific CHA hairpin probes, such that each miRNA resulted in distinct amplified photoluminescence (PL) decays that could be distinguished and quantified by TG PL intensity detection. Enzyme-free amplification in a separation-free assay format and the absence of autofluorescence background allowed for simple, specific, and sensitive detection of miR-21 and miR-20a with limits of detection down to 1.8 pM (250 amol). Reliable duplexed quantification of both miRNAs at low picomolar concentrations was confirmed by analyzing total RNA extracts from different colon and rectum tissues with single- and dual-target CHA-TG-FRET and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for comparison. These simple and multiplexed nucleic acid biomarker assays present a capable method for clinical diagnostics and biomolecular research.

Improving sensitivity for the targeted LC-MS/MS analysis of the peptide bradykinin using a design of experiments approach

The nonapeptide bradykinin is endogenously present only in low picomolar plasma concentrations, subsequently making reliable detection using liquid chromatography coupled to mass spectrometry (LC-MS/MS) challenging. Furthermore, non-specific adsorption during sample preparation and storage can lead to unpredictable peptide losses. To overcome these issues, a design of experiments (DoE) approach was applied, which consisted of a screening to identify impacting factors, optimisation and confirmation runs. On the one hand, different injection solvent compositions and sample collection materials were investigated in order to decrease non-specific adsorption. On the other hand, the addition of modifiers, which are known to enhance the signal intensity in LC-MS/MS, to the chromatographic mobile phase was examined. Polypropylene was the most suitable material among those investigated and resulted in a factor increase of 12.0 compared to LC-MS glass. The advantages of protein low-binding polypropylene versus standard polypropylene were fully compensated by the optimisation of the injection solvent. The latter substantially contributed to a decrease of non-specific adsorption of bradykinin. In this regard, bradykinin further benefitted from an organic fraction and a high amount of formic acid. Based on the DoE results, the final optimised injection solvent—consisting of 8.7% formic acid in 49.4/5.3/36.6 water/methanol/dimethyl sulfoxide (v/v/v)—was established. Furthermore, optimisation of the mobile phase composition yielded a signal intensity increase by a factor of 7.7. The transferability of the optimisation results conducted in neat solutions were successfully confirmed in human plasma. The applicability of this approach was further supported by the successful determination of low-abundance endogenous bradykinin levels in human plasma using LC-MS/MS.

Terbium-to-quantum dot Förster resonance energy transfer for homogeneous and sensitive detection of histone methyltransferase activity.

The development of rapid, simple, and versatile biosensors for monitoring the activity of histone modifying enzymes (HMEs) is needed for the improvement of diagnostic assays, screening of HME inhibitors, and a better understanding of HME kinetics in different environments. Nanoparticles can play an important role in this regard by improving or complementing currently available enzyme detection technologies. Here, we present the development and application of a homogeneous methyltransferase (SET7/9) assay based on time-gated Förster resonance energy transfer (TG-FRET) between terbium complexes (Tb) and luminescent semiconductor quantum dots (QDs). Specific binding of a Tb-antibody conjugate to a SET7/9-methylated Lys4 on a histone H3(1-21) peptide substrate attached to the QD surface resulted in efficient FRET and provided the mechanism for monitoring the SET7/9 activity. Two common peptide-QD attachment strategies (biotin-streptavidin and polyhistidine-mediated self-assembly), two different QD colors (625 and 705 nm), and enzyme sensing with post- or pre-assembled QD-peptide conjugates demonstrated the broad applicability of this assay design. Limits of detection in the low picomolar concentration range, high selectivity tested against non-specific antibodies, enzymes, and co-factors, determination of the inhibition constants of the SET7/9 inhibitors SAH and (R)-PFI-2, and analysis of the co-factor (SAM) concentration-dependent enzyme kinetics of SET7/9 which followed the Michaelis-Menten model highlighted the excellent performance of this TG-FRET HME activity assay.

Effective Targeting of PRAME-Positive Tumors with Bispecific T Cell-Engaging Receptor (TCER®) Molecules

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

Functional Titanium Carbide MXenes-Loaded Entropy-Driven RNA Explorer for Long Noncoding RNA PCA3 Imaging in Live Cells.

The visualization of the long noncoding RNA of prostate cancer gene 3 (lncRNA PCA3), a specific biomarker for androgen receptor-positive prostate cancer, in living cells not only directly reflects the gene expression and localization but also offers better insight into its roles in the pathological processes. Here, we loaded an entropy-driven RNA explorer (EDRE) on the TAT peptide-functionalized titanium carbide MXenes (Ti3C2-TAT) for the imaging of nuclear lncRNA PCA3 in live cells. The EDRE was condensed on the Ti3C2-TAT (Ti3C2-TAT@EDRE) by electrostatic interaction. Ti3C2-TAT@EDRE enables the entering of cells and release of TAT peptides and EDRE in the cytoplasm by the glutathione (GSH)-triggered cleavage of the disulfide bonds in Ti3C2-TAT. The released EDRE is delivered into the nucleus by the nucleus-targeted guidance of TAT peptides, and initiated by the target lncRNA PCA3, subsequently leading to the continuous accumulation of fluorescence signals. Consequently, fluorescence analysis of lncRNA PCA3 at low-picomolar concentrations in vitro as well as sensitive live cell imaging of lncRNA PCA3 in the nucleus of androgen receptor-positive LNCaP prostate cancer cells were achieved, providing a versatile strategy for the monitoring of nucleic acid biomarkers in the nucleus of living cells.