Sensitive LC-MS/MS quantification of unconjugated maytansinoid DM4 and its metabolite S-methyl-DM4 in human plasma.

Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.

Synthesis, characterization, and targeted chemotherapy of SCT200-linker-monomethyl auristatin E conjugates.

Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.

Population Pharmacokinetics of Trastuzumab Deruxtecan in Patients With HER2-Positive Breast Cancer and Other Solid Tumors.

Trastuzumab deruxtecan (DS-8201) is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate with a novel enzyme-cleavable linker, a topoisomerase I inhibitor payload, and a drug-to-antibody ratio of ≈ 8. We have characterized the population pharmacokinetics (PK) of trastuzumab deruxtecan and released drug (topoisomerase I inhibitor) in patients with HER2-positive breast cancer or other solid tumor malignancies. This analysis includes pooled data from five clinical studies with 639 patients. Trastuzumab deruxtecan doses ranged from 0.8 to 8.0 mg/kg every 3 weeks. Serum concentrations of trastuzumab deruxtecan and released drug were analyzed using a sequential two-step approach, with the nonlinear mixed-effects modeling methods. Covariate assessment was based upon stepwise forward-addition and backward-elimination process, followed by both univariate and multivariate analysis quantifying their impact on steady-state exposure of trastuzumab deruxtecan and released drug. A two-compartment model with linear elimination best described PK profiles of intact trastuzumab deruxtecan, while a one-compartment model with time-varying release-rate constant and linear elimination described released-drug PK profiles. Statistically significant covariates (country, tumor size, sex, formulation, age, body weight, albumin, total bilirubin, and aspartate aminotransferase) resulted in < 20% change in steady-state area under the concentration-time curve of trastuzumab deruxtecan and released drug, except for increased body weight (95th percentile, 86 kg) and decreased albumin (5th percentile, 31 g/L). Analysis of patients stratified by country, race, renal function, and hepatic function found no clinically meaningful differences in steady-state exposure of intact trastuzumab deruxtecan or released drug. Overall, results suggest that no dose adjustment based on tested covariates or in specific patient populations is warranted.

An Efficient Conjugation Approach for Coupling Drugs to Native Antibodies via the PtII Linker Lx for Improved Manufacturability of Antibody-Drug Conjugates.

The PtII linker [ethylenediamineplatinum(II)]2+ , coined Lx, has emerged as a novel non-conventional approach to antibody-drug conjugates (ADCs) and has shown its potential in preclinical in vitro and in vivo benchmark studies. A crucial improvement of the Lx conjugation reaction from initially <15 % to ca. 75-90 % conjugation efficiency is described, resulting from a systematic screening of all relevant reaction parameters. NaI, a strikingly simple inorganic salt additive, greatly improves the conjugation efficiency as well as the conjugation selectivity simply by exchanging the leaving chloride ligand on Cl-Lx-drug complexes (which are direct precursors for Lx-ADCs) for iodide, thus generating I-Lx-drug complexes as more reactive species. Using this iodide effect, we developed a general and highly practical conjugation procedure that is scalable: our lead Lx-ADC was produced on a 5 g scale with an outstanding conjugation efficiency of 89 %.

Antibody Co-Administration Can Improve Systemic and Local Distribution of Antibody-Drug Conjugates to Increase In Vivo Efficacy.

Several antibody-drug conjugates (ADC) showing strong clinical responses in solid tumors target high expression antigens (HER2, TROP2, Nectin-4, and folate receptor alpha/FRα). Highly expressed tumor antigens often have significant low-level expression in normal tissues, resulting in the potential for target-mediated drug disposition (TMDD) and increased clearance. However, ADCs often do not cross-react with normal tissue in animal models used to test efficacy (typically mice), and the impact of ADC binding to normal tissue antigens on tumor response remains unclear. An antibody that cross-reacts with human and murine FRα was generated and tested in an animal model where the antibody/ADC bind both human tumor FRα and mouse FRα in normal tissue. Previous work has demonstrated that a "carrier" dose of unconjugated antibody can improve the tumor penetration of ADCs with high expression target-antigens. A carrier dose was employed to study the impact on cross-reactive ADC clearance, distribution, and efficacy. Co-administration of unconjugated anti-FRα antibody with the ADC-improved efficacy, even in low expression models where co-administration normally lowers efficacy. By reducing target-antigen-mediated clearance in normal tissue, the co-administered antibody increased systemic exposure, improved tumor tissue penetration, reduced target-antigen-mediated uptake in normal tissue, and increased ADC efficacy. However, payload potency and tumor antigen saturation are also critical to efficacy, as shown with reduced efficacy using too high of a carrier dose. The judicious use of higher antibody doses, either through lower DAR or carrier doses, can improve the therapeutic window by increasing efficacy while lowering target-mediated toxicity in normal tissue.

Conjugating MMAE to a novel anti-HER2 antibody for selective targeted delivery.

OBJECTIVE: To investigate the target delivery properties of RC48-ADC, a novel antibody drug conjugate (ADC) comprising cytotoxic monomethyl auristatin E (MMAE) and an anti-human epidermal growth factor receptor 2 (HER2) antibody tethered via valine-citrulline linker, in vitro and in vivo. MATERIALS AND METHODS: Dissociation rate of MMAE from RC48-ADC was used as an estimate of its stability in serum. Cytotoxicity of the antibody and RC48-ADC towards multiple cell lines was measured. Subcellular distribution of the drug was determined by fluorescence imaging. The mechanism of lysosome targeting was verified. Endocytic pathways of RC48-ADC were assessed by the cellular fluorescence intensity of fluorescently-labelled drugs. Intracellular and extracellular distribution of MMAE was analysed after RC48-ADC or MMAE administration to characterize MMAE release. The serum and tumour concentration of MMAE was compared after tail-vein injection of RC48-ADC into tumour-bearing mice. RESULTS: RC48-ADC was highly stable in human serum. HER2-overexpressed cell line SK-BR-3 proliferation was stronger when suppressed by RC48-ADC than by the naked antibody. Both RC48-ADC and naked antibody were internalized via caveolae-mediated and clathrin-mediated endocytosis and concentrated in lysosomes. Higher HER2 expression was associated with enhanced uptake and intracellular release of conjugated MMAE; free MMAE could kill tumour cells via the bystander effect. Although serum RC48-ADC concentration was higher than that in tumours, exposure of MMAE in tumours was ~200 times higher than in serum, which rationalized the reduced toxicity of RC48-ADC. CONCLUSIONS: In vitro and in vivo experiments confirmed the targeted transport and release of RC48-ADC; it could selectively deliver MMAE to the targeted HER2-positive cell or tumour tissue, which could reduce off-target toxicity and enhance anti-tumour potency in humans.

Physiologically Based Pharmacokinetic Model-Informed Drug Development for Polatuzumab Vedotin: Label for Drug-Drug Interactions Without Dedicated Clinical Trials.

Model-informed drug development (MIDD) has become an important approach to improving clinical trial efficiency, optimizing drug dosing, and proposing drug labeling in the absence of dedicated clinical trials. For the first time, we developed a physiologically based pharmacokinetic (PBPK) model-based approach to assess CYP3A-mediated drug-drug interaction (DDI) risk for polatuzumab vedotin (Polivy), an anti-CD79b-vc-monomethyl auristatin E (MMAE) antibody-drug conjugate (ADC). The model was developed and verified using data from the existing clinical DDI study for brentuximab vedotin, a similar vc-MMAE ADC. Analogous to the brentuximab vedotin clinical study, polatuzumab vedotin at the proposed labeled dose was predicted to have a limited drug interaction potential with strong CYP3A inhibitor and inducer. Polatuzumab vedotin was also predicted to neither inhibit nor induce CYP3A. The present work demonstrated a high-impact application using a PBPK MIDD approach to predict the CYP3A-mediated DDI to enable drug labeling in the absence of any dedicated clinical DDI study. The key considerations for the PBPK report included in the Biologics License Application/Marketing Authorization Application submission, as well as the strategy and responses to address some of the critical and challenging questions from the health authorities following the submission are also discussed. Our experience and associated perspective using a PBPK approach to ultimately enable a drug interaction label claim for polatuzumab vedotin in lieu of a dedicated clinical DDI study, as well as the interactions with the regulatory agencies, further provides confidence in applying MIDD to accelerate the registration and approval of new drug therapies.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

A novel semi-mechanistic tumor growth fraction model for translation of preclinical efficacy of anti-glypican 3 antibody drug conjugate to human.

The growing fraction (GF) of tumor has been reported as one of the predictive markers of the efficacy of chemotherapeutics. Therefore, a semi-mechanistic model has been developed that describes tumor growth on the basis of cell cycle, allowing the incorporation of the GF of a tumor in pharmacokinetic/pharmacodynamic (PK/PD) modeling. Efficacy data of anti-glypican 3 (GPC3) antibody drug conjugate (ADC) in a hepatocellular carcinoma (HCC) patient derived xenograft (PDX) model was used for evaluation of this proposed model. Our model was able to describe the kinetics of growth inhibition of HCC PDX models following treatment with anti-GPC3 ADC remarkably well. The estimated tumurostatic concentrations were used in tandem with human PKs translated from cynomolgus monkey for prediction of the efficacious dose. The projected efficacious human dose of anti-GPC3 ADC was in the range 0.20-0.63 mg/kg for the Q3W dosing regimen, with a median dose of 0.50 mg/kg. This publication is the first step in evaluating the applicability of GF in PK/PD modeling of ADCs. The authors are hopeful that incorporation of GF will result in an improved translation of the preclinical efficacy of ADCs to clinical settings and thereby better prediction of the efficacious human dose.

T-DM1-induced thrombocytopenia in breast cancer patients: New perspectives.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) is overexpressed in 15-20% of patients with breast cancer. HER2 overexpression is the result of a genetic alteration and this marker is associated with poor clinical outcomes. HER2-targeted therapy can significantly improve the prognosis of patients with either early or advanced HER2-positive breast cancer. One such therapy is the antibody drug conjugate (ADC) trastuzumab emtansine (T-DM1), a combination of trastuzumab and the cytotoxic antimicrotubule agent DM1. After T-DM1 binds HER2, DM1 is subsequently released into the cell. T-DM1 is generally well tolerated and has a relatively low incidence of adverse events. However, there are clinical concerns regarding T-DM1-induced high-grade thrombocytopenia. METHODS: Here, we summarize the incidence of thrombocytopenia from several clinical trials and review experimental studies to explore the causes for T-DM1-induced thrombocytopenia. Progress in several other ADCs targeting HER2-positive breast cancer was also reviewed. CONCLUSIONS: We conclude that T-DM1 uptake by megakaryocytes occurs through either Fcγ receptor binding or through pinocytosis, and we suggest several methods through which these processes could be interrupted to potentially improve the clinical safety of T-DM1. More generally, we recommend that toxicity should be carefully addressed during the development of ADCs.

Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

A phase 1 study evaluating safety and pharmacokinetics of losatuxizumab vedotin (ABBV-221), an anti-EGFR antibody-drug conjugate carrying monomethyl auristatin E, in patients with solid tumors likely to overexpress EGFR.

Losatuxizumab vedotin (formerly ABBV-221) is a second-generation antibody-drug conjugate targeting epidermal growth factor receptor (EGFR). In this multicenter phase 1 study, eligible patients with EGFR-dependent solid tumors received losatuxizumab vedotin (3 + 3 design) intravenously at starting dose of 0.3 mg/kg over 3 h per 21-day cycle, with alternate dosing schedules utilized (2 weeks on/1 week off or weekly) to mitigate infusion reactions. Forty-five patients received ≥1 doses of losatuxizumab vedotin (13 colon, 6 non-small cell lung cancer, 5 head and neck [HNC], 5 glioblastoma multiforme, 2 breast, 14 other). Tumor samples were evaluated for EGFR protein expression by immunohistochemistry, EGFR and EGFR ligand mRNA expression by RNAseq, and results compared with outcome. Most common adverse events were infusion-related reaction (22/45; 49%) and fatigue (20/45; 44%). While most infusion reactions were grade ≤ 2, four patients experienced grade ≥3 infusion reactions. Several infusion reaction mitigation strategies were explored. Because of the high incidence of infusion reactions, the trial was stopped and the maximum tolerated dose was not reached. The last cleared dose: 6 mg/kg/cycle. Nineteen patients (42%) had stable disease; 4 remained on study >6 months. One HNC patient with increased levels of EGFR and EGFR ligands (amphiregulin, epiregulin) achieved a confirmed partial response. Pharmacokinetic analysis of losatuxizumab vedotin showed exposures appeared to be approximately dose-proportional. The high frequency of infusion reactions necessitated early closure of this trial. The detailed mitigation strategies used in this protocol for infusion-related reactions may provide beneficial information for trial design of agents with high infusion reaction rates.

An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics.

While antibody-drug conjugates (ADCs) are advancing through clinical testing and receiving new marketing approvals, improvements to the technology continue to be developed in both academic and industrial laboratories. Among the key ADC attributes that can be improved upon with new technology are their biodistribution and pharmacokinetic properties. During the course of ADC development, it has become apparent that conjugation of drugs to the surface of a monoclonal antibody can alter its physicochemical characteristics in a manner that results in increased nonspecific interactions and more rapid elimination from plasma. Researchers in the field have typically relied upon in vivo studies in preclinical models to understand how a particular ADC chemistry will impact these biological characteristics. In previous work, we described how animal studies have revealed a relationship between ADC hydrophobicity, pharmacokinetics, and nonspecific hepatic clearance, particularly by sinusoidal endothelium and Kupffer cells. Here, we describe a fluorescence-based assay using cultured Kupffer cells to recapitulate the nonspecific interactions that lead to ADC clearance in an in vitro setting with the aim of developing a tool for predicting the pharmacokinetics of novel ADC designs. Output from this assay has demonstrated an excellent correlation with plasma clearance for a series of closely related ADCs bearing discrete PEG chains of varying length and has proven useful in interrogating the mechanism of the interactions between ADCs and Kupffer cells.

First-in-Humans Imaging with 89Zr-Df-IAB22M2C Anti-CD8 Minibody in Patients with Solid Malignancies: Preliminary Pharmacokinetics, Biodistribution, and Lesion Targeting.

Immunotherapy is becoming the mainstay for treatment of a variety of malignancies, but only a subset of patients responds to treatment. Tumor-infiltrating CD8-positive (CD8+) T lymphocytes play a central role in antitumor immune responses. Noninvasive imaging of CD8+ T cells may provide new insights into the mechanisms of immunotherapy and potentially predict treatment response. We are studying the safety and utility of 89Zr-IAB22M2C, a radiolabeled minibody against CD8+ T cells, for targeted imaging of CD8+ T cells in patients with cancer. Methods: The initial dose escalation phase of this first-in-humans prospective study included 6 patients (melanoma, 1; lung, 4; hepatocellular carcinoma, 1). Patients received approximately 111 MBq (3 mCi) of 89Zr-IAB22M2C (at minibody mass doses of 0.2, 0.5, 1.0, 1.5, 5, or 10 mg) as a single dose, followed by PET/CT scans at approximately 1-2, 6-8, 24, 48, and 96-144 h after injection. Biodistribution in normal organs, lymph nodes, and lesions was evaluated. In addition, serum samples were obtained at approximately 5, 30, and 60 min and later at the times of imaging. Patients were monitored for safety during infusion and up to the last imaging time point. Results:89Zr-IAB22M2C infusion was well tolerated, with no immediate or delayed side effects observed after injection. Serum clearance was typically biexponential and dependent on the mass of minibody administered. Areas under the serum time-activity curve, normalized to administered activity, ranged from 1.3 h/L for 0.2 mg to 8.9 h/L for 10 mg. Biodistribution was dependent on the minibody mass administered. The highest uptake was always in spleen, followed by bone marrow. Liver uptake was more pronounced with higher minibody masses. Kidney uptake was typically low. Prominent uptake was seen in multiple normal lymph nodes as early as 2 h after injection, peaking by 24-48 h after injection. Uptake in tumor lesions was seen on imaging as early as 2 h after injection, with most 89Zr-IAB22M2C-positive lesions detectable by 24 h. Lesions were visualized early in patients receiving treatment, with SUV ranging from 5.85 to 22.8 in 6 target lesions. Conclusion:89Zr-IAB22M2C imaging is safe and has favorable kinetics for early imaging. Biodistribution suggests successful targeting of CD8+ T-cell-rich tissues. The observed targeting of tumor lesions suggests this may be informative for CD8+ T-cell accumulation within tumors. Further evaluation is under way.

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Cleavable linkers in antibody-drug conjugates.

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.

Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer.

Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.

Simultaneous quantification of total antibody and antibody-conjugated drug for XMT-1522 in human plasma using immunocapture-liquid chromatography/mass spectrometry.

XMT-1522, an antibody-drug conjugate (ADC) currently in Phase I clinical development, represents the first Dolaflexin®-based, cleavable ADC with a high drug-antibody ratio (DAR). In this work, a novel immunocapture LC-MS/MS method was successfully developed for the simultaneous quantification of both total antibody and cleavable antibody-conjugated drug auristatin F-hydroxypropylamide (AF-HPA) in human plasma. This method utilized microwave-assisted enzymatic digestion for the total antibody and chemical release of the drug from ADC on a 96-well based immunocapture sample preparation platform. The total antibody and the conjugated drug AF-HPA were separated and subsequently quantified concurrently by LC-MS/MS. The linear range of the standard curve for total antibody was from 50 to 5000 ng/mL and for AF-HPA was from 3.3 to 330 ng/mL. The linearities showed R2 ≥ 0.993 for total antibody and R2 ≥ 0.996 for AF-HPA, respectively. The intra- and inter-day precision and accuracy were well within 15%. The validated method, with the characteristics of high efficiency, great selectivity, free of carryover, short LC-MS/MS time (˜3.5 min) and low sample volume (20 µl), was successfully applied for analyzing Phase 1 cancer patient samples.

Characterization of Disulfide Bond Rebridged Fab-Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload.

We describe the characterization of antigen binding fragments (Fab)-drug conjugates prepared using a dual maleimide pyrrolobenzodiazepine dimer cytotoxic payload (SG3710). Pyrrolobenzodiazepine dimers, which are DNA cross-linkers, are a class of payloads used in antibody-drug conjugates (ADCs). SG3710 was designed to rebridge two adjacent cysteines, such as those that form the canonical interchain disulfide bond between the light and heavy chain in Fab fragments. The rebridging generated homogenous Fab conjugates, with a drug-to-Fab ratio of one, as demonstrated by the preparation of rebridged Fabs derived from the anti-HER2 trastuzumab antibody and from a negative control antibody both prepared using recombinant expression and papain digestion. The resulting anti-HER2 trastuzumab Fab-rebridged conjugate retained antigen binding, was stable in rat serum, and demonstrated potent and antigen-dependent cancer cell-killing ability. Disulfide rebridging with SG3710 is a generic approach to prepare Fab-pyrrolobenzodiazepine dimer conjugates, which does not require the Fabs to be engineered for conjugation. Thus, SG3710 offers a flexible and straightforward platform for the controlled assembly of pyrrolobenzodiazepine dimer conjugates from any Fab for oncology applications.

Method development of a novel PK assay for antibody-conjugated drug measurement of ADCs using peptide-linker drug analyte.

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.