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.

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.

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.

Antibody-Drug Conjugate Payloads; Study of Auristatin Derivatives.

Auristatins are important payloads used in antibody drug conjugates (ADCs), and the most well-known compound family member, monomethyl auristatin (MMAE), is used in two Food and Drug Administration (FDA)-approved ADCs, Adcetris® and Polivy®. Multiple other auristatin-based ADCs are currently being evaluated in human clinical trials and further studies on this class of molecule are underway by several academic and industrial research groups. Our group's main focus is to investigate the structure-activity relationships (SAR) of novel auristatins with the goal of applying these to next generation ADCs. Modifications of the auristatin backbone scaffold have been widely reported in the chemical literature focusing on the terminal subunits: P1 (N-terminus) and P5 (C-terminus). Our approach was to modulate the activity and hydrophilic character through modifications of the central subunits P2-P3-P4 and thorough SAR study on the P5 subunit. Novel hydrophilic auristatins were observed to have greater potency in vitro and displayed enhanced in vivo antitumor activity when conjugated via protease-cleavable linkers and delivered intracellularly. Analysis of ADC aggregation also indicated that novel hydrophilic payloads enabled the synthesis of high-drug-to-antibody ratio (DAR) ADCs that were resistant to aggregation. Modification of the central peptide subunits also resulted in auristatins with potent cytotoxic activity in vitro and these azide-modified auristatins contain a handle for linker attachment from the central portion of the auristatin backbone.

Comparison of Analytical Methods for Antibody-Drug Conjugates Produced by Chemical Site-Specific Conjugation: First-Generation AJICAP.

Antibody-drug conjugates (ADCs) have become a major class of oncology biopharmaceuticals. Traditional ADCs have a stochastic distribution of cytotoxic drugs attached at several different sites on the antibody. The heterogeneous nature of stochastic ADCs results in a complex compositional analysis. To improve on traditional ADC technology, we have developed a chemical conjugation platform termed "AJICAP" for the site-specific modification of native antibodies using a class of IgG Fc affinity reagents. Here we report further investigation focusing on several analyses of a first-generation AJICAP-ADC (Angew. Chem., Int. Ed. 2019, 58, 5592-5597). For drug-antibody ratio (DAR) determination, we examined and compared six different analytical methods. To the best of our knowledge, this is the first report of a comparison of analytical techniques to measure the DAR for ADCs produced by a site-specific technology such as AJICAP. Furthermore, a rapid analytical process for confirmation of the site selectivity of AJICAP conjugation was established by SEC-Q-TOF-MS. The analytical strategy reported here can be applied to the DAR determination of site-specific ADCs.

AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies.

The need for atom-precise biomolecule modification, and particularly the irreversible formation of covalent bonds to specific amino acids in proteins, has become an essential issue in the fields of pharmaceuticals and chemical biology. For example, antibody-drug conjugates (ADCs) are increasingly common entries into the clinical oncology pipeline. Herein, we report a new method of affinity peptide mediated regiodivergent functionalization (AJICAP™) that enables the synthesis of ADCs from native IgG antibodies. We succeeded in introducing thiol functional groups onto three lysine residues in IgGs using Fc affinity peptide reagents without antibody engineering. A cytotoxic molecule was then connected to the newly introduced thiol group, and both a surface plasmon resonance binding assay and in vivo xenograft mouse model results showed that the resulting ADC could selectively target and kill HER2-positive cells. Our strategy provides a new approach for constructing complex antibody-derived biomolecules.

Metabolism of an Oxime-Linked Antibody Drug Conjugate, AGS62P1, and Characterization of Its Identified Metabolite.

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).

Investigation of Hydrophilic Auristatin Derivatives for Use in Antibody Drug Conjugates.

Antibody drug conjugates offer a targeted cancer treatment for the delivery of potent cytotoxic drugs. Derivatives of the natural product dolastatin 10 containing pyridines and other basic amines were examined with the objective of determining if a more hydrophilic auristatin derivative would be potent enough for use as part of an ADC. This may be advantageous if a less hydrophobic drug makes a better ADC. A pyridine derivative, monomethyl auristatin PYE, showed the greatest potency when tested in vivo. While only a modest tumor growth inhibition was observed when the HCC1954 human breast cancer xenografts were treated with"non-cleavable" linker ADCs, tumor regression was seen when treated with an enzymatically degradable "cleavable" linker ADC when conjugated to trastuzumab. Based on these studies, monomethyl auristatin PYE shows promise for use as an ADC payload.

Synthesis and Evaluation of Dolastatin 10 Analogues Containing Heteroatoms on the Amino Acid Side Chains.

Synthetic analogues of the natural occurring dolastatin 10 are of great interest in cancer due to their potent in vitro activity and their uses as payloads in antibody drug conjugates (ADCs). Modification of the dolastatin 10 core scaffold has mainly focused on modifications of the P1, N-terminus, and P5, C-terminus, with minimal attention to the P2 subunit. In this paper we discuss the introduction of heteroatoms to the P2 side chain, which results in potent activity in vitro. The most active compounds contained azides in the P2 unit and required a phenylalanine-derived P5 subunit.

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.

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.

Oxidation of α-oxo-oximes to nitrile oxides with hypervalent iodine reagents.

Upon reaction with PhI(OAc)(2), α-oxo-aldoximes are oxidized to α-oxo-nitrile oxides, while α-oxo-ketoximes are converted into nitrile oxides via the oxidative cleavage of the carbonyl-imino σ bond. The nitrile oxides thus formed were trapped with norbornene or styrene in good yield. α,α'-Dioxo-ketoximes react less efficiently.

An overview of process development for antibody-drug conjugates produced by chemical conjugation technology.

Introduction: We discuss chemical conjugation strategies for antibody-drug conjugates (ADCs) from an industrial perspective and compare three promising chemical conjugation technologies to produce site-specific ADCs.Areas covered: Currently, nine ADCs are commercially approved and all are produced by chemical conjugation technology. However, seven of these ADCs contain a relatively broad drug distribution, potentially limiting their therapeutic indices. In 2019, the first site-specific ADC was launched on the market by Daiichi-Sankyo. This achievement, and an analysis of clinical trials over the last decade, indicates that current industrial interest in the ADC field is shifting toward site-specific conjugation technologies. From an industrial point of view, we aim to provide guidance regarding established conjugation methodologies that have already been applied to scale-up stages. With an emphasis on highly productive, scalable, and synthetic process robustness, conjugation methodologies for ADC production is discussed herein.Expert opinion: All three chemical conjugation technologies described in this review have various advantages and disadvantages, therefore drug developers can utilize these depending on their biological and/or protein targets. The future landscape of the ADC field is also discussed.

In-situ Reverse Phased HPLC Analysis of Intact Antibody-Drug Conjugates.

The field of oncology has recently seen an exponential growth in antibody-drug conjugates (ADCs) as a biopharmaceutical class with seven ADCs being launched onto the market in the last ten years. Despite the increase in the industrial research and development of these compounds, their structural complexity and heterogeneity continue to present various challenges regarding their analysis including reaction monitoring. Robust and simple reaction monitoring analysis are in demand in the view of at-line in-process monitoring, and can instill control, confidence and reliability in the ADC manufacturing process. Aiming at providing chromatographic methods for conjugation monitoring, we evaluated herein the potential of utilizing reverse phase HPLC analysis, without sample pretreatment, for characterization of traditional cysteine-based ADCs. This analysis can be used for estimation of drug antibody ratio (DAR), which has shown the same trends and results as other well-established HPLC techniques. This methodology was also applied to three ADCs derived from three different antibodies. Additionally, we analyzed unpurified ADC samples existing in a complex reaction matrix and separated ADC species and payload compounds. This investigation was conducted using three different ADCs based on different payloads. The results described herein indicate the potential application of this RP-HPLC methodology in reaction monitoring studies.

Physical characteristics comparison between maytansinoid-based and auristatin-based antibody-drug conjugates.

Aim: Direct analytical comparison of two major drug-linkers in the antibody-drug conjugate (ADC) field was conducted. Methods: Four different analytical methods [AlogP calculation, reverse phase (RP) high-performance liquid chromatography (HPLC; RP-HPLC), size exclusion chromatography HPLC (SEC-HPLC), and differential scanning calorimetry (DSC)] were tested for this comparison. Results: Maytansinoid-based ADCs showed less hydrophobicity than auristatin-based ADCs. Regardless of the drug-linker and drug-to-antibody ratios (DARs), the stability detected by DSC was decreased by conjugation. Conclusions: The cost and time-efficient analytical comparison described in this manuscript may be useful information for an initial characterization of ADCs prior to detailed biological studies.

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.

Application of Native Ion Exchange Mass Spectrometry to Intact and Subunit Analysis of Site-Specific Antibody-Drug Conjugates Produced by AJICAP First Generation Technology.

Antibody-drug conjugates (ADCs) are at the forefront of the next generation of oncology biopharmaceuticals. Conventional ADCs involve stochastic conjugation of the antibody to a cytotoxic drug, creating a highly heterogeneous product. The resulting stochastic distribution often leads to a narrow therapeutic index and makes it difficult to analyze the composition of heterogeneous ADCs. With the goal of overcoming these issues, we developed a site-specific conjugation technology, named AJICAP, for production of low heterogeneity ADCs. For analysis of these site-specific ADCs, we report herein strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS). Retention time reproducibility after SCX column equilibration enabled monitoring of important changes in product quality. SCX-UV-MS performed with MS-compatible mobile phases was conducted for intact native ADC analysis, allowing drug-antibody ratio characterization and charge variant characterization in single analysis. Furthermore, subunit analysis of the site-specific ADCs by native SCX-UV-MS confirmed the Fc site selectivity of ADCs generated by AJICAP conjugation.

A Purification Strategy Utilizing Hydrophobic Interaction Chromatography to Obtain Homogeneous Species from a Site-Specific Antibody Drug Conjugate Produced by AJICAP™ First Generation.

In recent years, site-specific antibody drug conjugates (ADC)s have been in great demand because they have an expanded therapeutic index compared with conventional ADCs. AJICAP™ technology is a chemical conjugation platform to obtain site-specific ADCs through the use of a class of Fc-affinity compounds. Promising results from early technology development studies led to further investigation of AJICAP™ ADC materials to obtain site-specific and homogeneous drug antibody ratio (DAR) ADCs. Here we report site-specific conjugation followed by a preparative hydrophobic interaction chromatography (HIC) purification strategy to obtain purified "DAR = 1.0" and "DAR = 2.0" AJICAP™ ADC materials. Optimization of the mobile phase conditions and resin achieved a high recovery rate. In vitro biological assay demonstrated the target selective activity for purified homogeneous DAR ADCs. These results indicate the ability of a HIC purification strategy to provide "DAR = 1.0" and "DAR = 2.0" AJICAP™ ADCs with considerable potency and target selectivity.

Proof of site-specificity of antibody-drug conjugates produced by chemical conjugation technology: AJICAP first generation.

Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.

Analytical Comparison of Antibody-drug Conjugates Based on Good Manufacturing Practice Strategies.

The production of antibody-drug conjugates (ADCs) has been in great demand in the field of cancer therapeutics. Although cysteine-based conjugation is the most common and well known process for producing ADCs, multiple analytical methods are required for accurate drug-antibody ratio (DAR) determination due to the heterogeneity of the ADCs. Here we report various analytical methods for DAR analysis of traditional cysteine-based ADCs; additionally, apply a good manufacturing practice (GMP) strategy to produce a four hundred milligram ADC batch for use in good laboratory practice (GLP) studies. The work described herein not only evaluates several analytical performances but also provides guidance for future phase appropriate ADC production while establishing a unique analytical strategy.