Targeted desialylation and cytolysis of tumour cells by fusing a sialidase to a bispecific T-cell engager.

Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.

Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells.

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Neutralizing Antibodies to SARS-CoV-2 Selected from a Human Antibody Library Constructed Decades Ago.

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.

A new immunochemical strategy for triple-negative breast cancer therapy.

Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these cancers is sorely needed. Antibody-drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of ß-lactam-derivatized drugs. Following conjugation to ß-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.

Selection of a picomolar antibody that targets CXCR2-mediated neutrophil activation and alleviates EAE symptoms.

Receptors and their ligands are important therapeutic targets for about one third of marketed drugs. Here, we describe an epitope-guided approach for selection of antibodies that modulate cellular signaling of targeted receptors. We chose CXC chemokine receptor 2 (CXCR2) in the G-protein coupled receptor superfamily as receptor and a CXCR2 N-terminal peptide for antibody selection. We obtain a highly selective, tight-binding antibody from a 1011-member antibody library using combinatorial enrichment. Structural and Hydrogen-Deuterium-Exchange mass spectrometry analyses demonstrate antibody interaction with an N-terminal region of CXCR2 that is part of the IL-8 epitope. The antibody strongly inhibits IL-8-induced and CXCR2-mediated neutrophil chemotaxis in vitro and alleviates hCXCR2-dependent experimental autoimmune encephalomyelitis symptoms in mice. As inappropriate neutrophil migration accompanies many diseases including inflammatory bowel disease, glomerulonephritis, allergic asthma, chronic obstructive pulmonary disease, and cancer, this antibody has potential for development as a therapeutic agent, akin to anti-TNF antibodies. However, an important difference here is that the antibody targets the chemokine receptor and competes with natural ligand, rather than targeting the ligand itself.

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism.

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.

DNA-Encoded Libraries: Hydrazide as a Pluripotent Precursor for On-DNA Synthesis of Various Azole Derivatives.

DNA-encoded combinatorial chemical library (DEL) technology, an approach that combines the power of genetics and chemistry, has emerged as an invaluable tool in drug discovery. Skeletal diversity plays a fundamental importance in DEL applications, and relies heavily on novel DNA-compatible chemical reactions. We report herein a phylogenic chemical transformation strategy using DNA-conjugated benzoyl hydrazine as a common versatile precursor in azole chemical expansion of DELs. DNA-compatible reactions deriving from the common benzoyl hydrazine precursor showed excellent functional group tolerance with exceptional efficiency in the synthesis of various azoles, including oxadiazoles, thiadiazoles, and triazoles, under mild reaction conditions. The phylogenic chemical transformation strategy provides DELs a facile way to expand into various unique chemical spaces with privileged scaffolds and pharmacophores.

Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications.

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.

Avidity-Based Selection of Tissue-Specific CAR-T Cells from a Combinatorial Cellular Library of CARs.

Using T-cell chimeric antigen receptors (CAR-T) to activate and redirect T cells to tumors expressing the cognate antigen represents a powerful approach in cancer therapy. However, normal tissues with low expression of tumor-associated antigens (TAAs) can be mistargeted, resulting in severe side effects. An approach using a collection of T cells expressing a diverse, 106-member combinatorial cellular library of CARs, in which members can be specifically enriched based on avidity for cell membrane antigens, is reported. Using CD38 as the target antigen, an efficient and effective selection of CARs specifically recognizing CD38+ tumor cells is demonstrated. These selected CAR-T's produce cytokines known to be associated with T cell activation in a CD38 expression-dependent manner. This avidity-based selection endows the engineered T cells with minimal off-tumor effects, while retaining robust antitumor efficacy both in vitro and in vivo. The described method may facilitate the application of CAR-T therapy to TAAs previously considered undruggable.

gem-Difluoromethylene Alkyne-Enabled Diverse C-H Functionalization and Application to the on-DNA Synthesis of Difluorinated Isocoumarins.

Using gem-difluoromethylene alkynes as effectors, unprecedented diverse C-H activation/[4+2] annulations of simple benzoic acids are reported. The chemodivergent reaction outcomes are well-tuned by Rh/Ir-catalyzed system; in the RhIII catalysis, 3-alkenyl-1H-isochromen-1-one and 3,4-dialkylideneisochroman-1-one skeletons are afforded in a solvent-dependent manner whereas difluoromethylene-substituted 1H-isochromen-1-ones are generated under the IrIII -catalyzed system. Mechanistic studies revealed that unusually double ß-F eliminations and fluorine effect-induced regioselective reductive elimination are independently involved to enable distinct reaction modes for divergent product formations. Besides, synthetic application in both the derivatization of obtained diene products and the on-DNA synthesis of DNA-tagged difluorinated isocoumarin have been demonstrated, which manifested great potential for synthetic utility of the developed protocols.

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.

Selection of a Full Agonist Combinatorial Antibody that Rescues Leptin Deficiency In Vivo.

Growth factor deficiency in adulthood constitutes a distinct clinical syndrome with significant morbidities including abnormal body composition, reduced energy, affective disturbances, dyslipidemia, and increased cardiovascular risk. Protein replacement therapies using recombinant proteins or enzymes represent the only approved treatment. Combinatorial antibodies have shown great promise as a new class of therapeutic molecules because they act as "mechanism-based antibodies" with both agonist and antagonist activities. Using leptin, a key hormone in energy metabolism, as an example, a function-guided approach is developed to select combinatorial antibodies with high potency and full agonist activity that substitute natural growth factors in vivo. The identified antibody shows identical biochemical properties and cellular profiles as leptin, and rescues leptin-deficiency in ob/ob mice. Remarkably, the antibody activates leptin receptors that are otherwise nonfunctional because of mutations (L372A and A409E). Combinatorial antibodies have significant advantages over recombinant proteins for chronical usage in terms of immunological tolerance and biological stability.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses.

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

Selection of Small Molecules that Bind to and Activate the Insulin Receptor from a DNA-Encoded Library of Natural Products.

Although insulin is a life-saving medicine, administration by daily injection remains problematic. Our goal was to exploit the power of DNA-encoded libraries to identify molecules with insulin-like activity but with the potential to be developed as oral drugs. Our strategy involved using a 104-member DNA-encoded library containing 160 Traditional Chinese Medicines (nDEL) to identify molecules that bind to and activate the insulin receptor. Importantly, we used the natural ligand, insulin, to liberate bound molecules. Using this selection method on our relatively small, but highly diverse, nDEL yielded a molecule capable of both binding to and activating the insulin receptor. Chemical analysis showed this molecule to be a polycyclic analog of the guanidine metformin, a known drug used to treat diabetes. By using our protocol with other, even larger, DELs we can expect to identify additional organic molecules capable of binding to and activating the insulin receptor.

A DNA-encoded library for the identification of natural product binders that modulate poly (ADP-ribose) polymerase 1, a validated anti-cancer target.

Natural products have been an invaluable source of drug discovery, but their targets remain largely unknown. Natural products enriched DNA-encoded chemical libraries (nDELs) empower the researchers to rapidly and economically screen numerous natural products against various protein targets, and therefore promote the elucidation of the molecular mechanisms. In this work, we used poly (ADP-ribose) polymerase 1 (PARP1), as an example to explore the usage of nDEL for the functional natural products selection. We used late-stage modification approach to label three positive binders with unique DNA barcodes, whose dissociation constants range from sub-micromolar to micromolar. The selection criterion was set up according to the enrichment of these controls. Five natural products selected by this criterion directly bind to PARP1 in SPR, among which luteolin exhibits the highest inhibitory activity against PARP1. Moreover, luteolin selectively induces accumulation of DNA double-strand breaks and G2/M phase arrest in BRCA-deficient cells. All the findings from these investigations on luteolin support that PARP1 inhibition is one of the mechanisms for its anti-cancer activity.

Reflections on DNA-encoded chemical libraries.

In this short Editorial, we reflect on certain milestones that have led to the development of DNA-Encoded Chemical Libraries, while also providing a glimpse to future challenges and opportunities.