Sortase A-mediated Ligation Research Articles

Chemoenzymatic Synthesis of a Rhamnose-Functionalized Bispecific Nanobody as a Bispecific Antibody Mimic for Cancer Immunotherapy.

Bispecific antibodies (BsAbs) are next-generation therapeutics for complex cancer treatment. Herein, we developed a dual-targeting non-IgG format of bsAbs by using a bispecific nanobody (bsNb) that can simultaneously target EGFR and HER2 on tumor cells. Site-specific modification of the anti-EGFR-HER2 bsNb was conducted using the rhamnose (Rha) hapten via sortase A-mediated ligation to reconstitute the missing crystallizable fragment (Fc) effector biological functions. Functionally similar to bsAbs, bsNb-Rha conjugates retained dual-targeting activity and exerted potent anticancer effects via the Fc-domain-mediated engagement of endogenous anti-Rha antibodies. Further, an optimized bsNb-Rha conjugate exhibited markedly improved pharmacokinetics and efficient inhibitory effects against xenograft tumor growth in vivo. Our strategy provides a general and cost-effective platform to generate a new bsAb format for cancer immunotherapy.

Sortase A-mediated cyclization of novel polycyclic RGD peptides for ανβ3 integrin targeting

Cyclic arginine-glycine-aspartic (RGD) peptides that specifically bind to integrin ανβ3 have been developed for drug delivery, tracers, and imaging for tumor diagnosis and treatment. Herein, a series of polycyclic RGD peptides containing dual, tri, and tetra rings were designed and synthesized through sortase A-mediated ligation. An in vitro test on cell adhesion inhibition indicated that the RGD peptide containing tricylic structure exhibited outstanding potency and selectivity for ανβ3 integrin.

Sortase A: A chemoenzymatic approach for the labeling of cell surfaces.

Sortase A, a transpeptidase enzyme is present in many Gram-positive bacteria and helps in the recruitment of the cell surface proteins. Over the last two decades, Sortase A has become an attractive tool for performing in vivo and in vitro ligations. Sortase A-mediated ligation has continuously been used for its specificity, robustness, and highly efficient nature. These properties make it a popular choice among protein engineers as well as researchers from different fields. In this review, we give an overview of Sortase A-mediated ligation of various molecules on the cell surfaces, which can have diverse applications in interdisciplinary fields.

Exendin 4-Hapten Conjugate Capable of Binding with Endogenous Antibodies for Peptide Half-life Extension and Exerting Long-Acting Hypoglycemic Activity.

Hapten-specific endogenous antibodies are naturally occurring antibodies present in human blood. Herein, we investigated a new strategy in which small-molecule haptens were utilized as naturally occurring antibody binders for peptide half-life extension. The glucagon-like peptide 1 receptor agonist exendin 4 was site-specifically functionalized with the dinitrophenyl (DNP) hapten at the C-terminus via sortase A-mediated ligation. The resulting Ex4-DNP conjugates retained GLP-1 receptor activation potency in vitro and had a similar in vivo acute glucose-lowering effect comparable to that of native Ex4. Pharmacokinetic studies and hypoglycemic duration tests demonstrated that the Ex4-DNP conjugates displayed significantly elongated half-lives and improved long-acting antidiabetic activity in the presence of endogenous anti-DNP antibodies. In chronic treatment studies, once-daily administration of optimal conjugate 7 demonstrated more beneficial effects without prominent toxicity compared with Ex4. This strategy provides a new approach and represents an alternative to the well-established peptide-Fc fusion strategy to improve the peptide half-life and the therapeutic efficacy.

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials. One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Specific Inhibition of Tumor Growth by T Cell Receptor–Drug Conjugates Targeting Intracellular Cancer-Testis Antigen NY-ESO-1/LAGE-1

Despite the significant therapeutic advances in T-cell immunotherapy, many malignancies remain unresponsive, which might be because of the negative regulation of T cells by the tumor microenvironment (TME). T cells discriminate tumor cells and normal cells through T-cell receptors (TCRs); therefore, we generated a novel type of TCR-drug conjugates (TDCs) by referring antibody-drug conjugations (ADCs) to overcome the effects of the TME on T cells while preserving the specificity of TCR for tumor recognition. We selected HLA-A2/NY-ESO-1157-165 (peptide NY-ESO-1157-165 in complex with human leukocyte antigen serotype HLA-A*02:01) as the antigen and the antigen-specific TCR (1G4113) as the carrier. By sortase A-mediated ligation, we obtained three NY-TCR-vcMMAEs and further studied their properties, antitumor activity, and toxicity in vitro and in vivo. We found that all the NY-TCR-vcMMAEs had high endocytosis efficiency and specifically killed HLA-A2/NY-ESO-1157-165 positive tumor cells. In xenograft models, one of the TDCs, NY-TCR-2M, was effectively and specifically distributed into tumor tissues and inhibited tumor growth without inducing obvious toxicity. Our results demonstrated that TCRs can be carriers of toxic payloads and that the TDCs thus formed can specifically inhibit tumor growth, neglecting the immune microenvironment.

Recombinant production, bioconjugation and membrane binding studies of Pn3a, a selective NaV1.7 inhibitor

Chronic pain is a common and often debilitating condition. Existing treatments are either inefficacious or associated with a wide range of side effects. The progress on developing safer and more effective analgesics has been slow, in large part due to our limited understanding of the physiological mechanisms underlying pain in different diseases. Generation and propagation of action potentials is a central component of pain sensation and voltage-gated sodium channels (NaVs) play a critical role in this process. In particular, the NaV subtype 1.7, has emerged as a promising universal target for the treatment of pain.Recently, a spider venom peptide, μ-TRTX-Pn3a, was found to be a highly selective inhibitor of NaV1.7. Here, we report the first recombinant expression method for Pn3a in a bacterial host, which provides an inexpensive route to production. Furthermore, we have developed a method for bio-conjugation of our recombinantly produced Pn3a via sortase A-mediated ligation, providing avenues for further pre-clinical development.We demonstrate how heterologous expression in bacteria enables facile isotope labelling of Pn3a, which allowed us to study the membrane binding properties of the peptide by high-resolution solution-state nuclear magnetic resonance (NMR) spectroscopy using a recently developed lipid nanodisc system. The heteronuclear NMR data indicate that the C-terminal region of the peptide undergoes a conformational change upon lipid binding. The membrane binding properties of Pn3a are further validated using isothermal titration calorimetry (ITC), which revealed that Pn3a binds to zwitterionic planar lipid bilayers with thermodynamics that are largely driven by enthalpic contributions.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Chemical Biology of Autophagy-Related Proteins With Posttranslational Modifications: From Chemical Synthesis to Biological Applications.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved lysosomal degradation pathway in all eukaryotic cells, which is critical for maintaining cell homeostasis. A series of autophagy-related (ATG) proteins are involved in the regulation of autophagy. The activities of ATG proteins are mainly modulated by posttranslational modifications (PTMs), such as phosphorylation, lipidation, acetylation, ubiquitination, and sumoylation. To tackle molecular mechanisms of autophagy, more and more researches are focusing on the roles of PTMs in regulation of the activity of ATG proteins and autophagy process. The protein ligation techniques have emerged as powerful tools for the chemical engineering of proteins with PTMs, and provided effective methods to elucidate the molecular mechanism and physiological significance of PTMs. Recently, several ATG proteins with PTM were prepared by protein ligation techniques such as native chemical ligation (NCL), expressed protein ligation (EPL), peptide hydrazide-based NCL, and Sortase A-mediated ligation (SML). More importantly, the synthesized ATG proteins are successfully used to probe the mechanism of autophagy. In this review, we summarize protein ligation techniques for the preparation of ATG proteins with PTMs. In addition, we highlight the biological applications of synthetic ATG proteins to probe the autophagy mechanism.

End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions.

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.

Novel Method for Preparation of Site-Specific, Stoichiometric-Controlled Dual Warhead Conjugate of FGF2 via Dimerization Employing Sortase A-Mediated Ligation.

Targeted therapies are rapidly evolving modalities of cancer treatment. The largest group of currently developed biopharmaceuticals is antibody-drug conjugates (ADCs). Here, we developed a new modular strategy for the generation of cytotoxic bioconjugates, containing a homodimer of targeting protein and two highly potent anticancer drugs with distinct mechanisms of action. Instead of antibody, we applied human fibroblast growth factor 2 (FGF2) as a targeting protein. We produced a conjugate of FGF2 with either monomethyl auristatin E (MMAE) or α-amanitin (αAMTN) as a cytotoxic agent and subsequently applied a sortase A-mediated ligation to obtain a dimeric conjugate containing both MMAE and αAMTN. The developed method ensures site-specific conjugation and a controlled drug-to-protein ratio. We validated our approach by demonstrating that dimeric dual warhead conjugate exhibits higher cytotoxic potency against fibroblast growth factor receptor-positive cell lines than single-warhead conjugates. Our modular technology can be applied to other targeting proteins or drugs and thus can be used for preparation of different bioconjugates.

Design and synthesis of rhamnose-modified exenatide conjugate by sortase A-mediated ligation

Exenatide modified with rhamnose as a hapten was designed and synthesized by Sortase A-mediated ligation. An Exenatide peptide analog comprised of 46 amino acids, including the sortase A recognition motif LPETG at the C-terminus, was synthesized by solid phase peptide synthesis method. A tri-glycine modified-rhamnose derivative was chemically synthesized in seven steps in good yield. The site-specific conjugation between them catalyzed by Sortase A completed in 3 h and the rhamnose-modified Exenatide conjugate was obtained after semi-preparative HPLC purification and characterized by MALDI-TOF MS. This method should be generally useful for the synthesis of other Exenatide analog for biological studies.

New potent and selective αvβ3 integrin ligands: Macrocyclic peptides containing RGD motif synthesized by sortase A-mediated ligation

Three 14-mer macrocyclic peptides 1–3 containing mono-, di- and tri-RGD structure motif were designed and synthesized by sortase A-mediated ligation in good yields. The results of in intro cell-based biological assays indicated that linear peptide 5 and macrocyclic peptide 1, containing di-RGD and mono-RGD motif respectively, showed remarkable potency and selectivity to αvβ3 integrin.

High yield synthesis of cyclic analogues of antibacterial peptides P-113 by Sortase A-mediated ligation and their conformation studies

P-113 is a fragment of natural occurring peptide Histatin 5 found in human saliva. This peptide exhibited broad spectrum of antibacterial and antifungal biological activities. In this study, bifunctional P-113 peptides 2–5 were designed as Sortase A substrates and synthesized by solid support peptide synthesis, where the N-terminus were equipped with glycine and its analogues, and C-terminus were extended with LPETGGS, respectively. Under Sortase A catalyzed condition, head to tail cyclization products 7–10 were afforded in yields from 76% to 93%. The conformation insights of linear peptides 2–5 and cyclic analogues 7–10 in aqueous buffers and in trifluroethanol (TFE) analyzed by circular dichroism (CD) suggested that α-helix structures were produced progressively in hydrophobic environment independent of the cyclization, which displayed the similar behavior as parent peptide P-113.

Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli.

We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.

A Concise Approach to Site-Specific Topological Protein-Poly(amino acid) Conjugates Enabled by in Situ-Generated Functionalities.

Controlling the topology of polymer-modified proteins has attracted growing interest. However, one of the main challenges in this field is the lack of efficient and site-specific methods for installing multiple bioorthogonal functionalities on substrate polymers. We report here an orchestrating strategy that provides easy access to various topological protein-poly(amino acid) (PAA) conjugates in high yields. This method features the in situ installation of two "chemical handles", including a thioester for native chemical ligation and a polyglycine nucleophile for sortase A-mediated ligation, at both ends of substrate PAAs. As a result, neither pre-functionalization of initiator or monomer units, nor post-polymerization modification of the resultant polymers, is necessary. Site-specific topological conjugates, particularly circular conjugates, can be conveniently synthesized under mild conditions from the functionalized PAAs. The biomedical utility of our method is demonstrated by the rapid and efficient generation of several therapeutic interferon-α conjugates, which exhibit significantly enhanced protease resistance and thermostability. Given the versatility of both PAAs and proteins, the method offers a convenient approach to producing libraries of conjugates for biological applications.

Twigged streptavidin polymer as a scaffold for protein assembly

Protein assemblies are an emerging tool that is finding many biological and bioengineering applications. We here propose a method for the site-specific assembly of proteins on a twigged streptavidin (SA) polymer using streptavidin as a functional scaffold. SA was genetically appended with a G tag (sortase A recognition sequence) and a Y tag (HRP recognition sequence) on its N- and C-termini, respectively, to provide G-SA-Y. G-SA-Y was polymerized using HPR-mediated tyrosine coupling, then fluorescent proteins were immobilized on the polymer by biotin-SA affinity and sortase A-mediated ligation. Fluorescence measurements showed that the proteins were immobilized in close proximity to each other. Hydrolyzing enzymes were also functionally assembled on the G-SA-Y polymer. The site-specific assembly of proteins on twigged SA polymer may find new applications in various biological and bioengineering fields.

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly.

Context driven biosensor assembly with modular targeting and detection moieties is gaining significant attentions. Although protein-based nanoparticles have emerged as an excellent platform for biosensor assembly, current strategies of decorating bionanoparticles with targeting and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. Herein, we report a highly modular post-translational modification approach for biosensor assembly based on sortase A-mediated ligation. This approach enables the simultaneous modifications of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA aptamer, and dye decoration. The resulting easy-purification platform offers a high degree of targeting and detection modularity with signal amplification. This flexibility is demonstrated for the detection of both immobilized antigens and cancer cells.

Sortase A-mediated multi-functionalization of protein nanoparticles.

We report here a new strategy to enable fast, covalent, and site-directed functionalization of protein nanoparticles using Sortase A-mediated ligation using functional proteins ranging from monomeric to large tetrameric structures. Easy purification of the modified E2 nanoparticles is achieved by functionalization with a thermo-responsive elastin-like-peptide. The resulting protein nanoparticles remained intact and active even after repeated phase transitions, suggesting their use in biocatalysis, biosensing, and imaging applications.