The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a ...therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Immunoaffinity (IA) LC-MS/MS pharmacokinetic (PK) assays are widely used in the field for antibody drug conjugates (ADCs) containing peptide linkers that are enzymatically cleavable, such as ...MC-ValCit-PAB. Conjugate PK assay strategies for these ADCs involve cleavage with cathepsin B or papain to release and measure the antibody-conjugated drug (acDrug) concentration. However, robust acDrug PK methods for disulfide-linked self-immolating ADCs are lacking as they are a different conjugation modality. We developed acDrug PK assays for next-generation disulfide-linked ADCs involving immunoaffinity capture, chemical cleavage, and LC-MS/MS. Disulfide-linked ADCs captured from plasma were chemically reduced at basic pH to release the linker-drug, followed by self-immolation to liberate the active drug, and quantified by MRM LC-MS/MS. Herein, we detail the development and optimization of this chemical cleavage acDrug PK assay, resulting in robust accuracy and precision (±20%). The conjugation site of the linker-drug on the antibody was found to affect the kinetics of drug release. Multiple biophysical and chemical characteristics, such as tertiary structure, fractional solvent accessibility, pK a of the conjugation site, surrounding residue’s pI, and electrostatic charge, may directly impact the drug release kinetics. Similar site-specific stability has been previously reported for ADCs in vivo. The assay development and qualification data for this original assay format are presented along with its application to multiple in vitro and in vivo studies across species.
Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs. They are designed to target tumor antigens selectively and offer the hope of cancer treatment without ...the debilitating side-effects of conventional therapies. The concept of ADCs is not new; however, development of these therapeutics is challenging and only recently are promising clinical data emerging. These challenges include ADC bioanalysis, such as quantifying in serum/plasma for PK studies and strategies for assessing immunogenicity. ADCs have complex molecular structures incorporating large- and small-molecule characteristics and require diverse analytical methods, including ligand-binding assays and MS-based methods. ADCs are typically mixtures with a range of drug-to-antibody ratios. Biotransformations in vivo can lead to additional changes in drug-to-antibody ratios resulting in dynamically changing mixtures. Thus, a standard calibration curve consisting of the reference standard may not be appropriate for quantification of analytes in vivo and represents a unique challenge. This paper will share our perspective on why ADC bioanalysis is so complex and describe the strategies and rationale that we have used for ADCs, with highlights of original data from a variety of nonclinical and clinical case studies. Our strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.
Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC ...following binding, internalization and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalized by ADC dose with unknown mechanism.
Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals.
To determine MMAE RBC partitioning, tritium labeled MMAE (
H-MMAE) was incubated in whole blood from mice, rats, monkeys and humans
, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (
RBC partitioning).
Our
data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration).
dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat.
These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed
between preclinical and clinical studies. These findings highlight the importance of fully characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.
Antibody–drug conjugates (ADCs) are designed to facilitate the targeted delivery of cytotoxic drugs to improve their tumor fighting effects and minimize systemic toxicity. However, efficacy and ...safety can potentially be compromised due to the release of conjugated drugs from the ADC with time while in circulation, resulting in changes in the drug-to-antibody ratio (DAR). Current understanding of this process is limited because existing methods such as immunoassays fail to distinguish ADCs with different DARs. Here we demonstrate a novel method with bead-based affinity capture and capillary liquid chromatography–mass spectrometry to allow direct measurement of drug release by quantifying DAR distributions of the ADC in plasma/serum. This method successfully identified individual intact conjugated antibody species produced due to drug loss from ADCs (e.g., an engineered site-specific anti-MUC16 THIOMAB–drug conjugate) and measured the corresponding DAR distributions in vitro and in vivo. Information obtained can provide insights into the mechanisms involved in drug loss and help to optimize ADC therapeutics. Other potential applications of the method may include characterization of posttranslational modifications, protein adducts, and immunogenicity.
Quantitative analysis of antibody–drug conjugates (ADCs) involves cleavage of ADCs into smaller analytes representing different components and subsequent measurements from multiple assays for a more ...comprehensive pharmacokinetic (PK) assessment. Multiple PK analytes including the drug remaining conjugated to the antibody (or antibody-conjugated drug, acDrug) and total antibody can be accessed simultaneously using a multiplex assay by proteolytic digestion of an ADC, if the sites of conjugation are homogeneous for an ADC and the linker drug is stable to proteases. Herein, a multiplexed immunoaffinity liquid chromatography-mass spectrometry (LC-MS)/MS PK assay is described involving immunoaffinity enrichment, enzymatic conversion of prodrug, trypsin digestion, and LC-MS/MS as applied to next-generation ADCs constructed from linker drugs bearing dimeric cyclopropabenzindole (CBI) payloads (duocarmycin analogues). The cytotoxic payload is chemically labile, requiring extensive optimization in sample preparation steps to stabilize the drug without ex vivo modification and to convert the prodrug into a single active form of the drug. The qualification data for this assay format showed that this approach provides robust acDrug and total antibody data and can be extended to ADCs with different monoclonal antibody frameworks and linker chemistries. Applications of this multiplexed assay to support preclinical studies are presented.
Ultrasound-guided percutaneous renal biopsy (PRB) stands as an important tool of diagnostic and prognostic value, besides its role in determining the best therapeutic option in some diseases. The ...advance in medicine over the past years made PRB safer and more feasible. This is an observational retrospective study in a tertiary referral center of pediatric nephrology and was conducted to determine both the indications and the histopathological findings of renal biopsies performed in pediatric patients. The retrospective review of 73 files searching for gender, age, indication for renal biopsy, and histopathological diagnosis of biopsy was done. The statistical analysis was done using Microsoft Excel Worksheet version 2010. The files of 73 cases were reviewed, of which three were excluded due to inadequate sample (success rate of 95.9%). The mean age was 6.9 years (standard deviation ±3.51) with a male-to-female ratio of 1.8:1. The main indication for PRB was nephrotic syndrome (NS) (40%) mainly steroid-resistant NS. Focal segmental glomerulosclerosis was found in most of the cases (46.4%) followed by minimal change disease (32.1%). Among secondary glomerulonephritis, lupus nephritis (LN) was the most common indication (15.7%). Class IV LN came at the top of the list (45.5%). Poststreptococcal GN patients were biopsied when rapidly progressive GN was suspected. Immunoglobulin A nephropathy was found in only 1.4%. Other PRB indications were hematuria (8.6%), unexplained acute (2.9%), or chronic renal failure (4.3%). Renal biopsy remains to be a mainstay diagnostic tool in pediatric nephrology. This study confirms the reliability of PRB as a diagnostic tool which can probably impact the management and hence improve the outcome. The findings in our patients align with findings from other centers and differ in others denoting that disease distribution can vary from one place to another.
Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an
method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes ...within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment,
/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.
To evaluate the clinical immunogenicity of eight antibody-drug conjugates (ADCs), multi-domain biotherapeutics that could theoretically pose a greater immunogenicity risk than monoclonal antibodies ...(mAbs) because they contain non-natural structural motifs.
Immunogenicity strategies and assays for these ADCs included those commonly used for conventional biotherapeutics with additional characterization. A tiered approach was adopted for testing Phase I and II clinical study samples with screening, confirmatory assays and additional domain characterization. Antidrug antibody incidences with these ADCs were within those reported for mAb biotherapeutics with no apparent impact on clinical outcomes.
These data suggest that the ADC hapten-like structure across these eight ADCs does not appear to increase patient immune responses beyond those generally observed for mAb biotherapeutics.
Pharmacokinetic analysis of antibody-drug conjugates (ADCs) requires characterization and quantification of both the antibody-conjugated cytotoxic drug molecule (acDrug) as well as the antibody ...vehicle, among other analytes, in order to assess the safety and efficacy of ADCs. Due to the complexity of biological matrices, immunoaffinity capture is widely used for enrichment of the biotherapeutic, followed by enzymatic or chemical release of the drug and LC-MS/MS analysis to provide the concentration of acDrug. This bioanalytical strategy has been used successfully with ADCs, but is limited to ADCs having cleavable linkers. Herein, we developed a sensitive and specific method that involved subjecting the ADC to tryptic digestion, and measured a peptide that included cysteine conjugated to the drug to provide quantification of acDrug. Using this method for a THIOMAB™ antibody-drug conjugate (TDC) conjugated to MMAE via a cleavable linker, valine–citrulline, we compared peptide-linker MMAE data from the new assay format with earlier MMAE data for acDrug. This showed that the new assay format provides robust acDrug as well as total antibody concentration to study in vitro stability of the TDC in multiple matrices and in vivo pharmacokinetic models of TDC in rat and mouse. The data from the two orthogonal modes of acDrug analysis showed good agreement with each other, allowing us to successfully quantify acDrug to study the stability in vitro and the pharmacokinetic parameters in vivo. This new assay strategy allows acDrug quantification for ADCs with non-cleavable linkers where the resulting acDrug analyte is a peptide-linker drug.
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