The unique reactivity of two sortase enzymes, SrtAstaph from Staphylococcus aureus and SrtAstrep from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtAstrep is used to label the protein’s C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtAstaph and LPXT derivatives. The generality of N-terminal labeling with SrtAstaph is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.
Selective incorporation of unnatural amino acids into proteins is a powerful tool for illuminating the principles of protein design. In particular, fluorinated amino acids have recently emerged as valuable building blocks for designing hyperstable protein folds, as well as directing highly specific protein-protein interactions. We review the collagen mimetic and coiled coil peptide systems that exemplify generalizable paradigms for future design. The unique electronic and phase properties of fluorocarbons are discussed, and protein synthesis using unnatural amino acids is briefly reviewed.
Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.antibodies | bioorthogonal | engineering | transacylation T he creation of protein-protein fusions, by genetic means, chemically, or enzymatically, has become an important tool to study cell biological and biochemical problems. Genetic fusions with fluorescent proteins are widely used to visualize their (sub)cellular localization in situ and in vivo (1). Coexpression of two orthogonally labeled chimeras allows the study of protein colocalization and dynamics of receptor dimerization. Moreover, protein fusions have been used to evaluate the biological relevance of otherwise transient protein complexes. Fusion or cross-linking of two or more of the interacting proteins can stabilize protein complexes and has been used to explore signaling and (hetero)dimerization of G-protein-coupled receptors (2, 3), chemokines, and cytokines (4-6).Besides being useful biochemical tools, chimeric proteins are also promising as treatment options for cancer, autoimmune diseases, lysosomal storage diseases, and brain disorders (7-10). Toxins have been conjugated to antibodies, growth factors, and cytokines as a means of delivering these payloads to malignant cells that express the counterstructures recognized by such fusion proteins, to kill tumor cells while minimizing collateral damage (10-12). Bispecific antibodies, prepared by fusing two singlechain variable fragments (scFV) of immunoglobulins, may combine an antigen-binding domain specific for a tumor cell with a CD3 receptor-binding domain specific for T cells (13). These compounds then allow the T cells to exert cytotoxic activity or cytokine release locally and so elicit the desired antitumor response. Finally, protein fusion strategies have been used to prepare structurally defined biomaterials (14).The production and purification of fusion proteins remains a biotechnological challenge. To obtain an active product, both domains of the chimera must adopt the native fold, without modification of residues and regions that are required for activity. The standard method to produce such proteins is by genetic fusion of the ORFs of the two proteins or protein fragments. Although recombinant expressi...
The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.
Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.
Tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADCs) is now a clinically validated approach for cancer treatment. In an attempt to improve the clinical success rate of ADCs, emphasis has been recently placed on the use of DNA-cross-linking pyrrolobenzodiazepine compounds as the payload. Despite promising early clinical results with this class of ADCs, doses achievable have been low due to systemic toxicity. Here we describe the development of a new class of potent DNA-interacting agents wherein changing the mechanism of action from a cross-linker to a DNA alkylator improves the tolerability of the ADC. ADCs containing the DNA alkylator displayed similar in vitro potency, but improved bystander killing and in vivo efficacy, compared to those of the cross-linker. Thus, the improved in vivo tolerability and anti-tumor activity achieved in rodent models with ADCs of the novel DNA alkylator could provide an efficacious, yet safer option for cancer treatment.on May 9, 2018.
The number of life-threatening fungal infections has risen in immunocompromised patients, and identification of the rules that govern an appropriate immune response is essential to develop better diagnostics and targeted therapeutics. The outer cell wall component on pathogenic fungi consists of β-1,3-glucan, and Dectin-1, a pattern recognition receptor present on the cell surface of innate immune cells, binds specifically to this carbohydrate. A barrier in understanding the exact immunological response to pathogen-derived carbohydrate epitopes is the presence of multiple types of carbohydrate moieties on fungal cell walls. To dissect the immunological mechanisms used to recognize pathogens, a system of “fungal like particles” was developed that consisted of polystyrene beads, which mimicked the three dimensional shape of the fungus, coated covalently with purified β-1,3-glucan derived from Saccharomyces cerevisiae. The morphology of the β-1,3-glucan layer was examined by immunofluorescence, flow cytometery, and immuno-transmission electron microscopy. The covalent linkages of the β-1,3-glucan to the polystyrene surface were stable after subjecting the beads to detergents. By pre-treating β-1,3-glucan beads with laminarinase, a specific β-1,3-gluconase, the reactivity of the anti-β-1,3-glucan antibody was abrogated in comparison to treatment with proteinase K indicating that the coating of these beads was predominantly β-1,3-glucan. TNF-α was also measured by stimulating bone-marrow derived macrophages with the β-1,3-glucan beads, and showed a dose dependent response compared to soluble β-glucan, insoluble β-1,3-glucan, uncoated beads, and soluble β-1,3-glucan mixed with uncoated beads. Finally, β-1,3-glucan beads were incubated with GFP-Dectin-1 expressing macrophages and imaged using confocal microscopy. β-1,3-beads were taken up within minutes and retained Dectin-1 recruitment to the phagosome as compared to uncoated beads. This data describes a unique fungal-like particle system that will permit immunologists to probe the critical steps in early recognition of pathogen-derived fungal carbohydrate antigens by innate immune cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.