Site-specific Chemical Conjugation Research Articles

Chemogenetic Evolution of Diversified Photoenzymes for Enantioselective [2 + 2] Cycloadditions in Whole Cells.

Artificial photoenzymes with novel catalytic modes not found in nature are in high demand; yet, they also present significant challenges in the field of biocatalysis. In this study, a chemogenetic modification strategy is developed to facilitate the rapid diversification of photoenzymes. This strategy integrates site-specific chemical conjugation of various artificial photosensitizers into natural protein cavities and the iterative mutagenesis in cell lysates. Through rounds of directed evolution, prominent visible-light-activatable photoenzyme variants were developed, featuring a thioxanthone chromophore. They successfully enabled the enantioselective [2 + 2] photocycloaddition of 2-carboxamide indoles, a class of UV-sensitive substrates that are traditionally challenging for known photoenzymes. Furthermore, the versatility of this photoenzyme is demonstrated in enantioselective whole-cell photobiocatalysis, enabling the efficient synthesis of enantioenriched cyclobutane-fused indoline tetracycles. These findings significantly expand the photophysical properties of artificial photoenzymes, a critical factor in enhancing their potential for harnessing excited-state reactivity in stereoselective transformations.

A single backbone amide modification method to achieve single site-specific chemical protein conjugation.

Efficient and precise chemical protein modification methods are highly sought after in biotechnology. However, chemically distinguishing a single site within a large protein is challenging. This study introduces a Copper Assisted Sequence-specific Conjugation Tag (CAST) method, enabling rapid (second order rate 8.1M-1s-1) and site-specific chemical modification of the protein backbone with pinpoint accuracy. The versatility of this method is demonstrated through the preparation of antibody-drug conjugates, showcasing high plasma stability and potent efficacy in both in vitro and in vivo settings. Thus, CAST emerges as an efficient and quantitative approach for attaching payloads to large, native proteins.

Chemical Amplification-Enabled Topological Modification of Nucleic Acid Aptamers for Enhanced Cancer-Targeted Theranostics.

Site-specific chemical conjugation has long been a challenging endeavor in the field of ligand-directed modification to produce homogeneous conjugates for precision medicine. Here, we develop a chemical amplification-enabled topological modification (Chem-ATM) methodology to establish a versatile platform for the programmable modification of nucleic acid aptamers with designated functionalities. Differing from conventional conjugation strategies, a three-dimensional artificial base is designed in Chem-ATM as a chemical amplifier, giving access to structurally and functionally diversified conjugation of aptamers, with precise control over loading capacity but in a sequence-independent manner. Meanwhile, the sp3 hybridized atom-containing amplifier enables planar-to-stereo conformational transformation of the entire conjugate, eliciting high steric hindrance against enzymatic degradation in complex biological environments. The versatility of Chem-ATM is successfully demonstrated by its delivery of anticancer drugs and imaging agents for enhanced therapy and high-contrast noninvasive tumor imaging in xenograft and orthotopic tumor models. This study offers a different perspective for ligand-directed chemical conjugation to enrich the molecular toolbox for bioimaging and drug development.

AJICAP Second-generation: Realizing Versatility and Robustness through Fc-Affinity Guided Chemical Conjugation Technology

Over the past decade, site-specific chemical conjugation has emerged as a promising strategy to produce antibody-drug conjugates (ADCs) in the field of oncology [1]. Daiichi-Sankyo’s chemical conjugation method, which uses high drug-to-antibody ratio (DAR) technology, was approved by the U.S. Food and Drug Administration as the first site-specific ADC (fam-trastuzumab-deruxtecan-nxki; Enhertu® | Daiichi Sankyo and AstraZeneca). However, owing to limitations in high-DAR technology and the potential need for DAR=2 production, the Ajinomoto group has been developing a chemical site-specific conjugation technology using Fc-affinity peptide reagents since 2019 [2]. This approach modifies a specific lysine in the Fc region of various antibodies such as IgG1, IgG2, and IgG4 to create homogeneous DAR=2 ADCs, as demonstrated in a proof-of-concept study.

Abstract 1850: A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability

Abstract Immunocytokines (IC) leverage orthogonal mechanisms of action in one molecule to induce potent antitumor immune responses. PD-1-targeting ICs are of particular interest since they harbor the multifunctional ability to selectively target antigen-experienced PD-1+ CD8+ T cells enriched in the tumor microenvironment (TME), release them from PD-(L)1 pathway inhibition, be retained within the TME, and simultaneously deliver potent cytokine receptor stimulation to the same T cell in cis (cis-signaling). Interleukin (IL-18) is a proinflammatory cytokine that stimulates both innate and adaptive immunity and generates potent antitumor activity mediated by both T effector and NK cells. Recent evidence indicates that a subset of tumor-infiltrated PD-1+ CD8+ T effector cells hallmarked by high expression of IL-18 receptor exhibits a superior cytotoxic and proliferative phenotype. Hence, we developed a PD1-IL18 IC to specifically target and activate intratumoral IL-18R-expressing PD-1+ CD8+ T cells. We engineered a conjugatable variant of human IL-18 with enhanced potency and significant resistance to IL-18 binding protein (IL-18BP) and utilized this enhanced IL-18 payload to create a PD1-IL18 IC (BPT567) via site-specific chemical conjugation to a defined lysine residue within the heavy chain of an anti-human PD-1 antibody (Ab). Conjugation did not affect the basic properties of the Ab as neither binding to PD-1 nor the interaction with the neonatal Fc receptor (FcRn) or Fcγ receptors were significantly impacted. Of note, the conjugation handle in our enhanced IL-18 variant was inserted at a site distinct from its N- or C-terminus to preserve the full potency and selectivity of the IL-18 payload. As a result, BPT567 exhibits increased potency and marked resistance to IL-18BP inhibition compared to wild-type IL-18. In vivo, BPT567 shows striking antitumor efficacy in multiple syngeneic mouse tumor models that is superior to responses induced by an anti-PD-1 Ab alone, a non-targeted IL-18 IC or the combination of both agents. Our evidence suggests that the strong in vivo efficacy observed is attributed to a preferential activation of PD-1+ tumor-infiltrated immune cells. The excellent tolerability and large therapeutic window observed in mice may be driven by less potent immune cell activation in the periphery. Citation Format: Jean-Philippe Carralot, Kea Martin, Rubén Alvarez Sanchez, Roy Meoded, Caoimhe Herr, Philipp Moosman, Arnaud Goepfert, Andrew Chi, Vijaya J. Pattabiraman, Bertolt Kreft. A first-in-class PD1-IL18 immunocytokine (BPT567) targets PD-1+ IL18R+ CD8+ T effector cells enriched in the tumor microenvironment and exhibits potent antitumor efficacy with excellent tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1850.

Efficient secretory production of proline/alanine/serine (PAS) biopolymers in Corynebacterium glutamicum yielding a monodisperse biological alternative to polyethylene glycol (PEG)

BackgroundPAS biopolymers are recombinant polypeptides comprising the small uncharged l-amino acids Pro, Ala and/or Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behaviour in physiological solution results in a strongly expanded hydrodynamic volume. Thus, apart from their use as fusion partner for biopharmaceuticals to achieve prolonged half-life in vivo, PAS biopolymers appear attractive as substitute for PEG—or other poorly degradable chemical polymers—in many areas. As a prerequisite for the wide application of PAS biopolymers at affordable cost, we have established their highly efficient biotechnological production in Corynebacterium glutamicum serving as a well characterized bacterial host organism.ResultsUsing the CspA signal sequence, we have secreted two representative PAS biopolymers as polypeptides with ~ 600 and ~ 1200 amino acid residues, respectively. Both PAS biopolymers were purified from the culture supernatant by means of a simple downstream process in a truly monodisperse state as evidenced by ESI–MS. Yields after purification were up to ≥ 4 g per liter culture, with potential for further increase by strain optimization as well as fermentation and bioprocess development. Beyond direct application as hydrocolloids or to exploit their rheological properties, such PAS biopolymers are suitable for site-specific chemical conjugation with pharmacologically active molecules via their unique terminal amino or carboxyl groups. To enable the specific activation of the carboxylate, without interference by the free amino group, we generated a blocked N-terminus for the PAS(1200) polypeptide simply by introducing an N-terminal Gln residue which, after processing of the signal peptide, was cyclised to a chemically inert pyroglutamyl group upon acid treatment. The fact that PAS biopolymers are genetically encoded offers further conjugation strategies via incorporation of amino acids with reactive side chains (e.g., Cys, Lys, Glu/Asp) at defined positions.ConclusionsOur new PAS expression platform using Corynex® technology opens the way to applications of PASylation® technology in multiple areas such as the pharmaceutical industry, cosmetics and food technology.

Abstract 4224: BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development

Abstract High-dose recombinant IL-2 (aldesleukin) is approved for treatment of advanced melanoma and renal cell carcinoma, however, major limitations restrict its therapeutic use. Wild-type IL-2 acts via binding to the medium-affinity IL-2 receptor βγ (IL2Rβγ) expressed in CD8+ T effector cells and NK cells. At the same time, its efficacy is dampened due to strong activation of regulatory T cells (Treg) expressing the high-affinity IL-2 receptor αβγ (IL2Rαβγ). Furthermore, binding to CD25/IL2Rα on endothelial cells and type 2 innate lymphoid cells is thought to be involved in the induction of severe toxicity including vascular leak syndrome (VLS). In addition, aldesleukin exhibits a very short half-life that, combined with its safety risks, requires a burdensome inpatient treatment schedule. We set out to rationally design a variant of human IL-2 that addresses and overcomes the major limitations of aldesleukin. Using our chemical protein synthesis technology, we introduced select modified amino acids including site-specific chemical conjugation handles to optimize the properties of IL-2 for cancer therapy while maintaining high homology to the wild-type IL-2 sequence. Bright Peak’s enhanced cytokine shows increased binding to CD122/IL2Rβ and does not interact with CD25/IL2Rα to improve safety and prevent the preferential activation of Tregs compared to CD8+ T effector cells. Site-specific conjugation to a 30 kDa PEG for half-life extension resulted in the generation of BPT-143, which is equipotent to aldesleukin in activating CD8+ T cells in vitro. In mice, BPT-143 induces a strong expansion of CD8+ T cells with only transient and minor effects on Tregs in vivo and exhibits improved PK properties allowing for a convenient dosing schedule. In the syngeneic CT26 tumor model, BPT-143 showed strong anti-tumor efficacy as a single agent as well as enhanced efficacy in combination with an anti-PD-1 antibody. BPT-143 induced a 55% complete response rate and, upon tumor re-challenge, all cured animals rejected CT26 tumor cells indicating the development of immunologic memory. In multiple-dose PK/PD studies in non-human primates (NHP), BPT-143 was well tolerated and induced robust and repeated expansion of CD8+ T cells, CD4+ conventional T cells and NK cells while showing only negligible effects on Tregs and eosinophils. Both in vivo efficacy studies in murine tumor models as well as PD effects observed in NHP indicate that BPT-143 has a best-in-class profile among “not-alpha” IL-2 compounds currently in development. IND-enabling studies are ongoing and a first in human trial is planned to start in 2022. Citation Format: Jean-Philippe Carralot, Rubén Alvarez Sanchez, Matilde Arévalo Ruiz, Magali Muller, Vijaya R. Pattabiraman, Bertolt Kreft. BPT-143: a fully synthetic alpha-dead IL-2 with a best-in-class preclinical pharmacodynamic and efficacy profile supporting first-in-human clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4224.

Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates.

To overcome a lack of selectivity during the chemical modification of native non-engineered antibodies, we have developed a technology platform termed "AJICAP" for the site-specific chemical conjugation of antibodies through the use of a class of IgG Fc-affinity reagents. To date, a limited number of antibody-drug conjugates (ADCs) have been synthesized via this approach, and no toxicological study was reported. Herein, we describe the compatibility and robustness of AJICAP technology, which enabled the synthesis of a wide variety of ADCs. A stability assessment of a thiol-modified antibody synthesized by AJICAP technology indicated no appreciable increase in aggregation or decomposition upon prolonged storage, indicating that the unexpectedly stable thiol intermediate has a great potential intermediate for payload or linker screening or large-scale manufacturing. Payload conjugation with this stable thiol intermediate generated several AJICAP-ADCs. In vivo xenograft studies indicated that the AJICAP-ADCs displayed significant tumor inhibition comparable to benchmark ADC Kadcyla. Furthermore, a rat pharmacokinetic analysis and toxicology study indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index, compared with stochastic conjugation technology. This is the first report of the therapeutic index estimation of site-specific ADCs produced by utilizing Fc affinity reagent conjugation. The described site-specific conjugation technology is a powerful platform to enable next-generation ADCs through reduced heterogeneity and enhanced therapeutic index.

Mammalian Expression and In Situ Biotinylation of Extracellular Protein Targets for Directed Evolution.

Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated in situ and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA–protein fusion. HER2 affibody–mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody–mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.

Engineering selective molecular tethers to enhance suboptimal drug properties

Small-molecule drugs are utilized in a wide variety of clinical applications, however, many of these drugs suffer from one or more suboptimal properties that can hinder its delivery or cellular action in vivo, or even shelf an otherwise biologically tolerable drug. While high-throughput screening provides a method to discover drugs with altered chemical properties, directly engineering small-molecule bioconjugates provides an opportunity to specifically modulate drug properties rather than sifting through large drug libraries with seemingly ‘random’ drug properties. Herein, we propose that selectively “tethering” a drug molecule to an additional group with favorable properties will improve the drug conjugate's overall properties, such as solubility. Specifically, we outlined the site-specific chemical conjugation of rapamycin (RAP) to an additional “high-affinity” group to increase the overall affinity the drug has for cyclodextrin-based polymers (pCD). By doing so, we found that RAP's affinity for pCD and RAP's window of delivery from pCD microparticles was tripled without sacrificing RAP's cellular action. This synthesis method was applied to the concept of “affinity” for pCD, but other prosthetic groups can be used similarly. This study displays potential for increasing drug delivery windows of small-molecule drugs in pCD systems for chronic drug therapies and introduces the idea of altering drug properties to tune polymer-drug interactions.

A novel site-specific chemical conjugation of IgG antibodies by affinity peptide for immunoassays.

Recently, there has been an increasing interest in site-specific modifications of antibodies used in immunoassays for disease diagnosis and as antibody therapeutics, such as antibody-drug conjugates. Previously, we established a site-specific chemical conjugation system using an IgG-Fc binding chemical conjugation affinity peptide (CCAP). CCAP could be used only for the modification of human IgG owing to the lack of affinity of CCAP to rodent IgG molecules. In this study, novel CCAP reagents are proposed, which can be used for both human and mouse IgG, based on the Staphylococcus aureus protein A domain-derived affinity peptides Z34C and Z33. Compared with the activity of a conventional randomly modified antibody, mouse IgG modified using this method had favourable features in two immunoassays, demonstrating the advantages of the proposed CCAP method in preserving antibody functionality during conjugation.

Engineering Selective Molecular Tethers to Enhance Suboptimal Drug Properties

Small-molecule drugs are utilized in a wide variety of clinical applications, however, many of these drugs suffer from one or more suboptimal property that can hinder its delivery or cellular action in vivo, or even shelf an otherwise biologically tolerable drug. While high-throughput screening provides a method to discover drugs with altered chemical properties, directly engineering small-molecule bioconjugates provides an opportunity to specifically modulate drug properties rather than sifting through large drug libraries with seemingly 'random' drug properties. Herein, we propose that selectively tethering a drug molecule to an additional group with favorable properties will improve the drug conjugate's overall properties, such as solubility. Specifically, we outlined the site-specific chemical conjugation of rapamycin (RAP) to an additional high-affinity group to increase the overall the drug has for cyclodextrin-based polymers (pCD). By doing so, we found that RAP's for pCD and RAP's window of delivery from pCD microparticles was tripled without sacrificing RAP's cellular action. This synthesis method was applied to the concept of affinity for pCD, but other prosthetic groups can be used similarly. This study displays potential for increasing drug delivery windows of small-molecule drugs in pCD systems for chronic drug therapies and introduces the idea of altering drug properties to tune polymer-drug interactions.

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues—which can be chemical conjugation sites—were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

Gram-Scale Antibody–Drug Conjugate Synthesis by Site-Specific Chemical Conjugation: AJICAP First Generation

Antibody–drug conjugates (ADCs) have become a major class of cancer biopharmaceuticals. Traditional ADCs have a stochastic distribution of cytotoxic drugs linked to an antibody at several different...

Good Manufacturing Practice Strategy for Antibody-Drug Conjugate Synthesis Using Site-Specific Chemical Conjugation: First-Generation AJICAP.

The development of antibody–drug conjugates (ADCs) is in great demand in the oncology field. With the goal of maximizing the therapeutic index, the conjugation technology to produce ADCs has been shifted to a site-specific manner; however, it is still challenging to establish robust and scalable synthetic processes. We have developed a chemical conjugation platform termed AJICAP for site-specific ADC synthesis using IgG Fc-affinity peptides. Here, we report the preparation of site-specific ADCs based on first-generation AJICAP technology for use in good laboratory practice studies. Analysis of the final ADC product was conducted using validated systems and good manufacturing practice. This work may not only prompt further biological studies of AJICAP-ADC but also establish a strategy to provide well-documented manufacturing data to enable new drug application filings (e.g., investigational new drug applications) for site-specific ADCs.

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as "magic bullets". These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.

Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format.

Artificially modified IgG molecules are increasingly utilized in industrial and clinical applications. In the present study, the method of chemical conjugation by affinity peptide (CCAP) for site-specific chemical modification has been developed by using a peptide that bound with high affinity to human IgG-Fc. This method enabled a rapid modification of a specific residue (Lys248 on Fc) in a one-step reaction under mild condition to form a stable amide bond between the peptide and Fc. The monovalent peptide-IgG conjugate not only maintained complete antigen binding but also bound to Fc receptors (FcRn, FcγRI, and FcγRIIIa), indicating that it is a suitable conjugate form that can be further developed into highly functional antibody therapeutics. CCAP was applied for the preparation of an antibody-drug conjugate and a bispecific antibody to demonstrate the usefulness of this method.

Interferon dimers: IFN-PEG-IFN

Increasingly complex proteins can be made by a recombinant chemical approach where proteins that can be made easily can be combined by site-specific chemical conjugation to form multifunctional or more active protein therapeutics. Protein dimers may display increased avidity for cell surface receptors. The increased size of protein dimers may also increase circulation times. Cytokines bind to cell surface receptors that dimerise, so much of the solvent accessible surface of a cytokine is involved in binding to its target. Interferon (IFN) homo-dimers (IFN-PEG-IFN) were prepared by two methods: site-specific bis-alkylation conjugation of PEG to the two thiols of a native disulphide or to two imidazoles on a histidine tag of two His8-tagged IFN (His8IFN). Several control conjugates were also prepared to assess the relative activity of these IFN homo-dimers. The His8IFN-PEG20-His8IFN obtained by histidine-specific conjugation displayed marginally greater in vitro antiviral activity compared to the IFN-PEG20-IFN homo-dimer obtained by disulphide re-bridging conjugation. This result is consistent with previous observations in which enhanced retention of activity was made possible by conjugation to an N-terminal His-tag on the IFN. Comparison of the antiviral and antiproliferative activities of the two IFN homo-dimers prepared by disulphide re-bridging conjugation indicated that IFN-PEG10-IFN was more biologically active than IFN-PEG20-IFN. This result suggests that the size of PEG may influence the antiviral activity of IFN-PEG-IFN homo-dimers.

Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes.

The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene \u2013 methyltetrazine reactions

BackgroundFas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem.ResultsA procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab’ domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain.ConclusionsThe present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.