Bystander killing by anti-PTPRZ1 CAR-T cells using additional tumor cell lines and scFv

Primer pairs for CAR-T cell phenotyping by qPCR

Peptide synthesis is central to biomedical research, yet automated flow synthesis typically requires custom-built instrumentation, limiting accessibility beyond specialized laboratories. Here we demonstrate that a standard analytical HPLC can be repurposed for fully automated flow-based peptide synthesis. By packing a short guard column with resin and programming reagent injections through the autosampler, we achieved efficient coupling and deprotection cycles under temperature-controlled flow using T-BEC/Oxyma activation. This setup exploits the precise flow control, heating, and in-line UV detection intrinsic to HPLC systems, enabling real-time monitoring of Fmoc deprotection. Using this approach, we synthesized peptides up to 30 residues, including GLP-1 and bivalirudin, with comparable yields and purity to conventional solid-phase synthesis. No significant epimerization was observed under optimized conditions, and crude bivalirudin displayed thrombin inhibition activity identical to purified reference material. This work introduces a simple, safe, and economical platform for automated peptide synthesis with the advent of high temperature flow accessible to any laboratory equipped with an HPLC.

Anti-PTPRZ1 CAR-T cell effector function after incubation with the Ge1302_PTPRZ1-KI cells

In vivo anti-tumor activity of anti-PTPRZ1 CAR-T cells

Chemical ligation is an essential tool for constructing complex biomolecular architectures. To accelerate reaction discovery, a one-pot multi-substrate screening (OPMSS) platform was developed, combining peptide nucleic acid (PNA) tagging with direct MALDI analysis. This approach enables the simultaneous evaluation of multiple substrate pairs in a single pot without the need for chromatographic separation. Short PNA tags promote a uniform combinatorial pairing of substrates while the neutral polyamide backbone facilitates MALDI analysis to allow direct readout of ligated products as predominantly singly charged ions. Using this system, we readily detected established ligations, including Huisgen cycloaddition and amide bond formation, validating the platform in pilot screens pairing 8 × 8 substrates (64 possible combinations). Applying the method to discovery-mode screening of 13 × 11 substrates under visible-light photocatalytic conditions identified a previously unexplored ligation between alkyl azides and alkenes, consistent with pathways involving aminyl radicals or aminium radical cations. This work demonstrates the potential of OPMSS with PNA tagging as a practical and discovery-oriented approach for identifying new ligation reactions directly from complex mixtures.

Use of Jurkat triple reporter cells to assess CAR tonic signaling and specificity

Antibody-drug conjugates enable highly specific delivery of potent cytotoxics to biomarker-expressing cells. In parallel, advances in DNA circuitry and DNA-protein conjugates have allowed programmable integration of molecular inputs and signal amplification via hybridization chain reactions (HCRs). Here we present a system using affibody-DNA and aptamer-DNA conjugates to execute a Boolean logic operation on cell-surface biomarkers, resulting in amplified payload delivery using an HCR of DNA-drug conjugates. Proximity-induced assembly of the biomarker binders generates the initiator that triggers an HCR. The resulting assembly undergoes endocytosis, enabling controlled payload release of drugs conjugated to the DNA with cathepsin-cleavable linkers. We show that DNA-drug conjugates achieve targeted delivery with >100-fold amplification relative to the input biomarkers using fluorescence quantifications. We also identify payloads that strongly influence delivery efficiency and demonstrate delivery of different drug combinations. Finally, we show that biomarker-triggered HCRs can recruit generic antibodies. This modular technology enables tailored combinations of biomarker inputs and drug outputs toward more precise and personalized treatment.

Bystander killing by 471_28z CAR-T cells depends on soluble mediators and does not affect macrophages

Expression of effector/memory and activation/exhaustion markers by CAR-T cells in pre- vs. post-thawing samples

DNA-encoded library technologies have emerged as a powerful platform to rapidly screen for binders to a protein of interest. These technologies are underpinned by the ability to encode a rich diversity of small molecules. While large libraries are accessible by cycles of mix and split synthesis, libraries based on single chemistries tend to be redundant. Furthermore, the quality of libraries generally decreases with the number of synthetic transformations performed in its synthesis. An alternative approach is to use hybridization to program the combinatorial assembly of fragment pairs onto a library of DNA templates. A broad molecular diversity is more easily sampled since it arises from the pairing of diverse fragments. Upon identification of productive fragment pairs, a focused library covalently linking the fragments is prepared. This focused library includes linker of different length and geometry and offers the opportunity to enrich the selected fragment set with close neighbors. Herein we describe detailed protocols to covalently link diverse fragments and screen fragment-based libraries using commercially available microarray platform.

PTPRZ1 expression at the mRNA level according to different GBM clinicopathologic features

Antibody-drug conjugates (ADCs) offer the potential to deliver potent cytotoxics with unprecedented specificity with respect to a given biomarker. In parallel, development in DNA circuitry and DNA-protein conjugates provide opportunities to design systems that integrate inputs and compute outputs with the potential of amplified responses through hybridization chain reactions (HCRs). Herein, we report a system based on affinity miniproteins (affibodies)-DNA conjugates and aptamer-DNA conjugates to target specific cells via biomarker recognition, yielding a logic-gated response. We demonstrate that this system can be used to trigger HCRs conditionally either on the presence of two biomarkers or based on the density of a single biomarker. Additionally, we show that receptor-mediated uptake of the HCR-generated assemblies can be used to release a combination of payloads. This mechanism successfully achieves the targeted delivery of payloads through a cleavable linker in DNA-Drug conjugates (DDCs), in analogy to ADCs with an added amplification reaching above 100-fold. Crucially, we find that the nature of the drug in DDC is critical for the efficiency of this delivery and that a combination of drugs can be administered simultaneously. Finally, we show that biomarker-triggered HCRs can also be used to recruit generic antibodies.

Affinity proteins based on a three-helix bundle (affibodies, alphabodies, and computationally de novo designed ones) have been shown to be a general platform to discover binders with properties reminiscent of antibodies, combining high target specificity with affinities reaching well below the nanomolar. Herein, we report a strategy, coined self-assembled proteomimetic (SAP), to mimic such three-helix bundle architecture with a hybridization-enforced two-helix coiled coil that is obtained by templated native chemical ligation (T-NCL) of PNA-peptide conjugates. This SAP strategy stands out by its synthetic accessibility, reducing the length on the longest synthetic peptide to less than 30 amino acids which is readily attainable by standard SPPS methodologies. We show that the T-NCL dramatically accelerates the ligation, enabling this chemistry to proceed in a combinatorial fashion at low micromolar concentrations. We demonstrate that small combinatorial libraries of SAPs can be prepared in one operation and used directly in affinity selections against a target of interest with an LC-MS analysis of the fittest binders. Moreover, we show that the underlying design paradigm is functional for SAPs based on structurally distinct three-helix peptides aimed at different therapeutic targets, namely HER2 and spike's RBD, reaching picomolar affinities. We further illustrate that the affinity of the SAP can be allosterically regulated using a toehold displacement of the hybridizing PNAs to disrupt the coiled coil stabilization. Finally, we show that an RBD-targeting SAP effectively inhibits viral entry of SARS-CoV-2 with an IC50 of 2.8 nM.

Pleckstrin homology (PH) domains are structural motifs critical for cellular processes, such as signal transduction and cytoskeletal organization. Due to their involvement in various diseases, PH domains are promising therapeutic targets, yet their highly charged and hydrophobic binding sites are not ideal for traditional small drugs. In this study, we designed a DNA-encoded library (DEL) mimicking phospholipids to identify novel modulators targeting PH domains with uncharted chemical properties. Screening against several PH domains led to the discovery of 2DII, a small molecule that selectively binds to mSin1PH. This compound can modulate mTORC2 activity by impairing mTORC2's membrane interactions, resulting in reduced AKT1 phosphorylation. A micromapping via Dexter energy transfer based on 2DII bearing an iridium catalyst (2DII-Ir), along with a biotin-diazirine small molecule was used for target identification by proteomics, which confirmed mSin1 as the primary intracellular target of 2DII, demonstrating its potential for selective mTORC2 pathway modulation. These findings introduce a novel strategy for targeting PH domains and provide a foundation for the development of therapeutic interventions that modulate PH-domain-dependent signaling pathways.

DNA-encoded libraries connect the phenotypes of synthetic molecules to a DNA barcode; however, most libraries do not tap into the potential of Darwinian evolution. Herein, we report a DNA-templated synthesis (DTS) architecture to make peptides that are stabilized into α-helical conformations via head-to-tail supramolecular cyclization. Using a pilot library targeting MDM2, we show that repeated screening can amplify a binder from the lowest abundance in the library to a ranking that correlates to binding affinity. The study also highlights the need to design libraries such that the chemistry avoids biases from the heterogeneous yield in DTS.

Abstract The great success of chimeric antigen receptor (CAR) T-cell therapy in the treatment of patients with B-cell malignancies has prompted its translation to solid tumors. In the case of glioblastoma (GBM), clinical trials have shown modest efficacy, but efforts to develop more effective anti-GBM CAR T cells are ongoing. In this study, we selected protein tyrosine phosphatase receptor type Z (PTPRZ1) as a target for GBM treatment. We isolated six anti-human PTPRZ1 single-chain variable fragments from a human phage display library and produced second-generation CAR T cells in an RNA format. Patient-derived GBM PTPRZ1–knockin cell lines were used to select the CAR construct that showed high cytotoxicity while consistently displaying high CAR expression (471_28z). CAR T cells incorporating 471_28z were able to release IFNγ, IL2, TNFα, granzyme B, IL17A, IL6, and soluble FasL and displayed low tonic signaling. Additionally, they maintained an effector memory phenotype after in vitro killing. In addition, 471_28z CAR T cells displayed strong bystander killing against PTPRZ1-negative cell lines after preactivation by PTPRZ1-positive tumor cells but did not kill antigen-negative nontumor cells. In an orthotopic xenograft tumor model using NOD/SCIDγ mice, a single dose of anti-PTPRZ1 CAR T cells significantly delayed tumor growth. Taken together, these results validate PTPRZ1 as a GBM target and prompt the clinical translation of anti-PTPRZ1 CAR T cells.

Herein, we detail an extension of our research on the synthesis of a small library of furanoheliangolides and the characterization of the covalent interaction between goyazensolide and IPO5. Using a build-couple-pair strategy, we assembled a small library of germacrene-type lactones and diversified them into eight groups of structurally different analogues. The germacrene lactones were synthesized using Sonogashira coupling and Barbier-type macrocyclization, while the furanoheliangolides were further elaborated through gold-catalyzed transannulation followed by esterification. This synthetic approach enabled the generation of a goyazensolide alkyne-tagged cellular probe, which was used to identify the selective binding between goyazensolide and the oncoprotein importin-5 (IPO5). Mass spectrometry analysis of the proteolytic digest from the reaction between the goyazensolide probe and a recombinant IPO5 indicated a covalent engagement at Cys560 of IPO5, which was confirmed by site-directed mutagenesis.

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.