Antibody Modification via Lipoic Acid Ligase A-Mediated Site-Specific Labeling.

Enzymatic modification, particularly utilizing lipoic acid ligase (LplA), has emerged as a transformative approach in biopharmaceuticals, enabling precise and site-specific protein modifications. This review delves into the innovative applications of LplA in antibody modifications, including the creation of antibody-drug conjugates (ADCs) and the advancement of tag-free conjugation techniques. LplA's ability to facilitate the incorporation of bioorthogonal groups and its adaptability to various substrates underscores its versatility. Key developments include the successful generation of dual-labeled antibodies and the application of LplA in modifying antibody fragments. Additionally, the review explores the potential for LplA to enhance the therapeutic efficacy of ADCs through improved drug-to-antibody ratios and site-specific payload attachment. The implications of these advancements are significant, suggesting that LplA-mediated modifications could lead to more effective and targeted antibody-based therapies. This review aims to provide a comprehensive overview of LplA's role in expanding the possibilities of enzymatic conjugation, setting the stage for future research and clinical applications.

Exo-Cleavable Linkers: Enhanced Stability and Therapeutic Efficacy in Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) combine cytotoxic payloads with monoclonal antibodies through chemical linkers. Finding linkers that both enhance circulatory stability and enable effective tumor payload release remains a challenge. The conventional valine-citrulline (Val-Cit) linker is associated with several inherent drawbacks, including hydrophobicity-induced aggregation, a limited drug-antibody ratio (DAR), and premature payload release. This study introduces an exolinker approach, repositioning the cleavable peptide linker at the exo position of the p-aminobenzylcarbamate moiety, as an advancement over conventional linear linkers. This design, which incorporates hydrophilic glutamic acid, addresses the limitations of the Val-Cit platform and improves the ADC in vivo profiles. In vitro and in vivo evaluations showed that exolinker ADCs reduced premature payload release, increased drug-to-antibody ratios, and avoided significant aggregation, even with hydrophobic payloads. Furthermore, the payload remained stably attached to the ADC even in the presence of enzymes like carboxylesterases and human neutrophil elastase, indicating the potential for a favorable safety profile.

Spatiotemporal Control of Protein Refolding through Flash-Change Reaction Conditions.

Recombinant protein production is an essential aspect of biopharmaceutical manufacturing, with Escherichia coli serving as a primary host organism. Protein refolding is vital for protein production; however, conventional refolding methods face challenges such as scale-up limitations and difficulties in controlling protein conformational changes on a millisecond scale. In this study, we demonstrate the novel application of flow microreactors (FMR) in controlling protein conformational changes on a millisecond scale, enabling efficient refolding processes and opening up new avenues in the science of FMR technology. FMR technology has been primarily employed for small-molecule synthesis, but our novel approach successfully expands its application to protein refolding, offering precise control of the buffer pH and solvent content. Using interleukin-6 as a model, the system yielded an impressive 96% pure refolded protein and allowed for gram-scale production. This FMR system allows flash changes in the reaction conditions, effectively circumventing protein aggregation during refolding. To the best of our knowledge, this is the first study to use FMR for protein refolding, which offers a more efficient and scalable method for protein production. The study results highlight the utility of the FMR as a high-throughput screening tool for streamlined scale-up and emphasize the importance of understanding and controlling intermediates in the refolding process. The FMR technique offers a promising approach for enhancing protein refolding efficiency and has demonstrated its potential in streamlining the process from laboratory-scale research to industrial-scale production, making it a game-changing technology in the field.

AJICAP-M: Traceless Affinity Peptide Mediated Conjugation Technology for Site-Selective Antibody-Drug Conjugate Synthesis.

A traceless site-selective conjugation method, "AJICAP-M", was developed for native antibodies at sites using Fc-affinity peptides, focusing on Lys248 or Lys288. It produces antibody-drug conjugates (ADCs) with consistent drug-to-antibody ratios, enhanced stability, and simplified manufacturing. Comparative in vivo assessment demonstrated AJICAP-M's superior stability over traditional ADCs. This technology has been successfully applied to continuous-flow manufacturing, marking the first achievement in site-selective ADC production. This manuscript outlines AJICAP-M's methodology and its effectiveness in ADC production.

Engineering CD3/CD137 Dual Specificity into a DLL3-Targeted T-Cell Engager Enhances T-Cell Infiltration and Efficacy against Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.

Tag-free protein modification by lipoate ligase A: exploring substrate tolerance.

This study delves into the functional intricacies of lipoate ligase A (LplA), an enzyme showing great promise in bioconjugation due to its unique capacity for introducing azido groups into proteins without requiring a genetic tag. We aimed to enhance the understanding of LplA's functionality, particularly its substrate tolerance and the reliability of various analytical techniques. A pivotal aspect of our approach was incorporating azido groups into a range of proteins, followed by the addition of the fluorescent molecule Cy3 via click chemistry. Analysis of fluorescent intensity in the altered proteins indicated varying degrees of conjugation. Additionally, phenyl resin-based RP-HPLC facilitated effective separation of modified proteins, unmodified proteins, and remaining fluorescent tags post-separation. SASA analysis provided insights into conjugation trends, guiding the identification of proteins amenable to LplA's tag-free modification. Our findings demonstrate LplA's broad substrate tolerability for protein modification.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification.

The concept of tag-free protein modification has attracted considerable interest in chemical biology because of its flexible and straightforward reaction process. In 2021, a groundbreaking approach using lipoate ligase A (LplA) for tag-free enzymatic modification of antibodies was unveiled, demonstrating its potential for the generation of precise antibody conjugates. In this study, to further explore LplA-mediated antibody-drug conjugate (ADC) synthesis, we performed initial biological evaluations of ADCs synthesized using LplA. Using the anti-HER2 antibody trastuzumab, we introduced octanoic acid azide using LplA and subsequently obtained an ADC using click chemistry with the drug DBCO-VC-PAB-MMAE. The bioactivity of the synthesized anti-HER2-ADC was evaluated using HER2-positive SKBR-3 and HER2-negative MCF7 cells. Its toxicity and selectivity were found to be comparable to those of the FDA-approved Kadcyla. In addition, a stability study involving rat and human plasma demonstrated the stability of the LplA-mediated ADC. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. These preliminary in vitro evaluations suggested that LplA-derived ADCs can have considerable pharmaceutical potential. Our results can set the stage for further in vivo evaluations and safety assessments. We suggest that the integration of tag-free LplA methods into the production of ADCs can offer a novel and promising approach for biopharmaceutical manufacturing.

Bispecific Antibodies Produced via Chemical Site-Specific Conjugation Technology: AJICAP Second-Generation.

Bispecific antibodies (BisAbs) are biotherapeutics that amalgamate the specificities of two distinct antibodies into one molecule, however, their engineering requires genetic modification and remains time-consuming. Therefore, we used AJICAP second-generation technology, which drives the production of site-specific conjugation without genetic modification requirements, to generate BisAbs. Using haloketone chemistry as an alternative to maleimide chemistry, we successfully produced site-specific antibody conjugates. Pharmacokinetic studies revealed that the haloketone-based antibody conjugate was stable in the rat plasma. The resultant BisAbs were rigorously evaluated, and surface plasmon resonance measurements and flow cytometry analyses confirmed that the antigen binding remained intact. Additionally, the affinity for the neonatal Fc receptor (FcRn) was retained after conjugation. Further cytotoxicity evaluation emphasized the pronounced activity of the generated BisAbs. This novel approach introduces a fully chemical, site-specific strategy capable of producing BisAbs, heralding a new era in the field of biotherapeutics.

Novel formats of antibody conjugates: recent advances in payload diversity, conjugation, and linker chemistry.

INTRODUCTION: In the field of bioconjugates, the focus on antibody - drug conjugates (ADCs) with novel payloads beyond the traditional categories of potent cytotoxic agents is increasing. These innovative ADCs exhibit various molecular formats, ranging from small-molecule payloads, such as immune agonists and proteolytic agents, to macromolecular payloads, such as oligonucleotides and proteins. AREAS COVERED: This review offers an in-depth exploration of unconventional strategies for designing conjugates with novel mechanisms of action and notable examples of approaches that show promising prospects. Representative examples of novel format payloads and their classification, attributes, and appropriate conjugation techniques are discussed in detail. EXPERT OPINION: The existing basic technologies used to manufacture ADCs can be directly applied to synthesize novel formatted conjugates. However, a wide variety of new payloads require the creation of customized technologies adapted to the unique characteristics of these payloads. Consequently, fundamental technologies, such as conjugation methods aimed at achieving high drug - antibody ratios and developing stable crosslinkers, are likely to become increasingly important research areas in the future.

Analytical studies on the conjugation site specificity of trastuzumab modified by Escherichia coli lipoate ligase A: multiple-enzyme digestion approach for peptide mapping.

Tag-free protein modification has received considerable attention in the field of chemical biology owing to the versatility and simplicity of the reaction sequence. In 2021, a novel tag-free enzymatic modification of antibodies utilizing lipoate ligase A (LplA) was reported to reveal its potential in the production of site-specific antibody conjugates. Primary peptide mapping analysis revealed the biased site specificity of antibodies modified by LplA; however, quantitative analysis remains challenging because of the complicated heterogeneity derived from biased selective modification. In an effort to further understand the site occupancy of LplA-modified antibodies, this study employed numerous unconventional techniques and strategies. Optimization of HPLC conditions and utilization of enzymes such as trypsin, Glu-C, and chymotrypsin significantly increased sequence data coverage. The transition from traditional spectral counting to a more accurate peak area-based label-free quantification helped better analyze peptide modification levels. The results obtained indicate that LplA-induced modifications are specific lysines, particularly the light chain Lys188/190 site, which have an increased modification rate compared to chemically induced modifications. This study not only contributes to the understanding of peptide modification, but also presents an improved methodology that promises to stimulate further research in this field.

AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugate Production.

The site-directed chemical conjugation of antibodies remains an area of great interest and active efforts within the antibody-drug conjugate (ADC) community. We previously reported a unique site modification using a class of immunoglobulin-G (IgG) Fc-affinity reagents to establish a versatile, streamlined, and site-selective conjugation of native antibodies to enhance the therapeutic index of the resultant ADCs. This methodology, termed "AJICAP", successfully modified Lys248 of native antibodies to produce site-specific ADC with a wider therapeutic index than the Food and Drug Administration-approved ADC, Kadcyla. However, the long reaction sequences, including the reduction-oxidation (redox) treatment, increased the aggregation level. In this manuscript, we aimed to present an updated Fc-affinity-mediated site-specific conjugation technology named "AJICAP second generation" without redox treatment utilizing a "one-pot" antibody modification reaction. The stability of Fc affinity reagents was improved owing to structural optimization, enabling the production of various ADCs without aggregation. In addition to Lys248 conjugation, Lys288 conjugated ADCs with homogeneous drug-to-antibody ratio of 2 were produced using different Fc affinity peptide reagent possessing a proper spacer linkage. These two conjugation technologies were used to produce over 20 ADCs from several combinations of antibodies and drug linkers. The in vivo profile of Lys248 and Lys288 conjugated ADCs was also compared. Furthermore, nontraditional ADC production, such as antibody-protein conjugates and antibody-oligonucleotide conjugates, were achieved. These results strongly indicate that this Fc affinity conjugation approach is a promising strategy for manufacturing site-specific antibody conjugates without antibody engineering.

Biological Evaluation of Maytansinoid-Based Site-Specific Antibody-Drug Conjugate Produced by Fully Chemical Conjugation Approach: AJICAP®.

BACKGROUND: Trastuzumab-emtansine (T-DM1, commercial name: Kadcyla) is well-known antibody-drug conjugate (ADC) and was first approved for human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. This molecular format consisting of trastuzumab and maytansinoid payload (emtansine) is very simple, however, T-DM1 has wide heterogeneity due to non-specific conjugation, lowering its therapeutic index (TI). METHODS: To overcome this issue during the chemical modification of the random conjugation approach to generate T-DM1, we developed a novel chemical conjugation technology termed "AJICAP®" for modification of antibodies in site-specific manner by IgG Fc-affinity peptide based reagents. RESULTS: In this study, we compared site-specific maytansinoid-based ADCs synthesized by AJICAP and T-DM1 in rat safety studies. The results indicated an increase in the maximum tolerated dose, demonstrating an expansion of the AJICAP-ADC therapeutic index compared with that of commercially available T-DM1. Gram scale preparation of this AJICAP-ADC and the initial stability study are also described. CONCLUSIONS: Trastuzumab-AJICAP-maytansinoid produced by this unique chemical conjugation methodology showed higher stability and tolerability than commercially available T-DM1.

Biological evaluation for anti-inflammatory effect of africane-type sesquiterpenoids.

Africane-type sesquiterpenoids are a unique tricyclic carbon architecture sesquiterpenoid isolated as natural products. Δ9(15) -africanene has been reported to exhibit anti-inflammatory activity for carrageenan-induced rat foot edema. In this study, we reported structure-activity relationship study of africane-type sesquiterpenoids and found that some africane-type sesquiterpenoid analogs and their synthetic intermediate showed potent anti-inflammatory activity. To identify the mode of action of africane-type sesquiterpenoids and their synthetic intermediate, we evaluated the anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. Treatment with the africane-type compounds and their synthetic intermediate suppressed LPS-induced expressions of Cox-2 protein and mRNAs of the inflammatory cytokines IL-1ß and IL-6 at the concentrations that did not affect cell viability. Interestingly, although these africane-type compounds and their synthetic intermediate suppressed the pro-inflammatory cytokines' expressions, the compounds did not modulate NF-κB activation. These results suggest that the africane-type compounds and their synthetic intermediate are anti-inflammatory compounds that suppress the expression of LPS-induced inflammatory mediators independently of NF-κB activation.

The oncogene-dependent resistance to reprogramming unveils cancer therapeutic targets.

The resistance to transcription factor-mediated reprogramming into pluripotent stem cells is one of the distinctive features of cancer cells. Here we dissect the profiles of reprogramming factor binding and the subsequent transcriptional response in cancer cells to reveal its underlying mechanisms. Using clear cell sarcomas (CCSs), we show that the driver oncogene EWS/ATF1 misdirects the reprogramming factors to cancer-specific enhancers and thereby impairs the transcriptional response toward pluripotency that is otherwise provoked. Sensitization to the reprogramming cue is observed in other cancer types when the corresponding oncogenic signals are pharmacologically inhibited. Exploiting this oncogene dependence of the transcriptional "stiffness," we identify mTOR signaling pathways downstream of EWS/ATF1 and discover that inhibiting mTOR activity substantially attenuates the propagation of CCS cells in vitro and in vivo. Our results demonstrate that the early transcriptional response to cell fate perturbations can be a faithful readout to identify effective therapeutics targets in cancer cells.

Scalable Syntheses and Biological Evaluation of Africane-Type Sesquiterpenoids.

Practical total syntheses of africane-type sesquiterpenoids were realized by reexamination of a divergent strategy employing optimized three-component coupling followed by ring-closing metathesis and substrate-controlled cyclopropanation. This sequential eight-step conversion provided Δ9(15) -africanene, a common bicyclo[5.3.0]decane intermediate for the syntheses of africane derivatives, in more than twice the yield as in the previous approach. The scalability and robustness of this improved synthetic route were confirmed by gram-scale preparation of Δ9(15) -africanene. In vitro cell-based assays of the synthesized africane-type sesquiterpenoids disclosed that ester-incorporating derivatives showed cytotoxic activity against HeLa cells. The effect of relative and absolute configuration of africane-9,15-diol monoacetates on the cytotoxicity against HeLa cells was also investigated.

Current approaches for the purification of antibody-drug conjugates.

In the past two decades, antibody-drug conjugates have gained increasing attention because they expand the therapeutic index when compared with that of traditional chemotherapies. Antibody-drug conjugates are highly complex structures consisting of antibodies covalently conjugated with small-molecule cytotoxic drugs. The complex structure of antibody-drug conjugates makes chemistry, manufacturing, and control difficult. In contrast to antibody production, distinct purification methods following conjugation of antibodies with drug-linkers are required for the manufacturing. For process development of antibody drug conjugates, the drug-to-antibody ratio, free drug-linkers, and aggregates are critical quality attributes that must be strictly controlled and removed by appropriate purification techniques. In this review, features of various purification methods used to purify antibody drug conjugates are described and evaluated. The future landscape of the antibody-conjugates field is also discussed briefly.

Recent Advances in Drug-Antibody Ratio Determination of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are biopharmaceuticals produced by chemically linking small molecules (payloads) to antibodies that possess specific affinity for the target cell. The ADCs currently on the commercially market are the result of a stochastic conjugation of highly-potent payloads to multiple sites on the monoclonal antibody, resulting in a heterogeneous drug-antibody ratio (DAR) and drug distribution. The heterogeneity inherent to ADCs not produced site-specifically may not only be detrimental to the quality of the drug but also is less-desirable from the perspective of regulatory science. An ideal method or unified approach used to measure the DAR for ADCs, a critical aspect of their analysis and characterization, has not yet been established in the ADC field and remains an often-challenging issue for bioanalytical chemists. In this review we describe, compare, and evaluate the characteristics of various DAR determination methods for ADCs featuring recently reported technologies. The future landscape of bioconjugate DAR analysis is also discussed.

Lipoate-acid ligase a modification of native antibody: Synthesis and conjugation site analysis.

Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis.

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.

Chromatographic analysis of site-specific antibody-drug conjugates produced by AJICAP first-generation technology using a recombinant FcγIIIa receptor-ligand affinity column.

Commercially approved conventional antibody-drug conjugates (ADCs) are produced as heterogeneous mixtures containing a stochastic distribution of payloads decorating the antibody molecules resulting in decreased efficacy and thus lowering their therapeutic index. Control of the DAR and conjugation site in the development of next-generation ADCs is believed to assist in increasing the therapeutic index of these targeted biologics leading to overall enhanced clinical efficacy and reduced toxicity. A chemical site-specific conjugation technology termed AJICAP® allows ADC developers to control both the location and quantity of the payload conjugation to an antibody. Furthermore, this simplified ADC composition enables a streamlined chemical analysis. Here we report the chromatographic separation of site-specific ADCs produced by AJICAP® technology using an analytical affinity chromatography HPLC column containing a recombinant FcγIIIa receptor-ligand immobilized on a non-porous polymer resin (NPR). These HPLC analyses provided visually clear chromatogram results reflecting the heterogeneity of each ADC. The affinity strength was also measured by biolayer interferometry (BLI) and predicted by molecular structure analysis. The results indicate that AJICAP® technology is a promising solution to link hydrophobic payloads to antibodies without compromising antibody receptor function. This study also shows that FcγIIIa-NPR column can be used to characterize site-specific conjugated ADCs compared to ADCs synthesized using conventional methods.