Preclinical characterization of XB002, an anti-tissue factor antibody-drug conjugate for the treatment of solid tumors.

Tissue factor (TF) is overexpressed in various cancers, where its expression is generally associated with poor disease outcomes. XB002 is an anti-TF antibody-drug conjugate designed to deliver a cytotoxic payload to TF-expressing tumors while minimizing adverse events related to disruption of TF function, notably bleeding. XB002 is composed of a zovodotin linker-payload conjugated to a monoclonal antibody (clone 25A3) that binds to TF with high affinity (KD = 0.

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.

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.

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.

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.

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.

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.

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.

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.

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

: 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.: 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.

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.

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.

Synthesis and Biological Evaluation of Antibody Drug Conjugates Based on an Antibody Expression System: Conamax.

Antibody production for ADCs (or in general) is commonly performed by CHO-based platforms and limited by volumetric productivity, expensive downstream purification, and extended optimization timelines. The Conamax platform is a novel microbial-based protein production and secretion system. A suite of synthetic biology tools have enabled high volumetric productivity (>1 g/L/d) and glycoengineering to produce simple and consistent human-like post-translational modifications.

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.

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.

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.

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.

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.

Synthesis and Evaluation of Linear and Macrocyclic Dolastatin 10 Analogues Containing Pyrrolidine Ring Modifications.

Because of their potent cytotoxic activity, members of the auristatin family (synthetic analogues of the naturally occurring dolastatin 10) have remained a target of significant research, most notably in the context of antibody drug conjugate payloads. Typically, modifications of the backbone scaffold of dolastatin 10 have focused on variations of the N-terminal (P1) and C-terminal (P5) subunits. Scant attention has been paid thus far to the P4 subunit in the scientific literature.

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.

Modulation of Macropinocytosis-Mediated Internalization Decreases Ocular Toxicity of Antibody-Drug Conjugates.

AGS-16C3F is an antibody-drug conjugate (ADC) against ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) containing the mcMMAF linker-payload currently in development for treatment of metastatic renal cell carcinoma. AGS-16C3F and other ADCs have been reported to cause ocular toxicity in patients by unknown mechanisms. To investigate this toxicity, we developed an assay using human corneal epithelial cells (HCEC) and show that HCECs internalized AGS-16C3F and other ADCs by macropinocytosis, causing inhibition of cell proliferation.

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.

A Potential Mechanism for ADC-Induced Neutropenia: Role of Neutrophils in Their Own Demise.

Neutropenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC) and we aimed to elucidate the potential mechanism of this toxicity. To investigate whether ADCs affect neutrophil production from bone marrow, an assay was developed in which hematopoietic stem cells (HSC) were differentiated to neutrophils. Several antibodies against targets absent in HSCs and neutrophils were conjugated to MMAE via a cleavable valine-citrulline linker (vcMMAE-ADC) or MMAF via a noncleavable maleimidocaproyl linker (mcMMAF-ADC), and their cytotoxicity was tested in the neutrophil differentiation assay.

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.

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.