Shrikant Deshpande

Production of human monoclonal antibody in eggs of chimeric chickens

The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell–derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

An integrated multiplatform bioanalytical strategy for antibody–drug conjugates: a novel case study

BACKGROUND The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Novel detection of DNA-alkylated adducts of antibody–drug conjugates with potentially unique preclinical and biomarker applications

BACKGROUND MDX-1203 is an antibody-drug conjugate (ADC) currently in clinical trials for the treatment of renal carcinoma. The active ingredient of MDX-1203 is a DNA minor groove-binding cytotoxic drug that forms a covalently linked adduct with an adenine base. Formation of this adenine adduct prevents DNA replication, thus triggering cell death. RESULTS A method has been developed to successfully isolate, identify and quantitate the adenine adduct using LC-MS/MS. The method is highly useful to validate the mode of action of this class of ADCs. Additionally, we have demonstrated that this method could potentially be utilized to assess the efficacy of the ADC in in vitro studies by measuring the amount of adenine adduct in various cells expressing the antigen. CONCLUSION Upon validation, this method could serve as an invaluable tool to evaluate compounds in preclinical in vivo models and in utilizing the DNA adduct as a potential biomarker.

Antibody–drug conjugate bioanalysis using LB-LC–MS/MS hybrid assays: strategies, methodology and correlation to ligand-binding assays

BACKGROUND Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. METHODS A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. RESULTS & CONCLUSION Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.

Abstract 2587: In vitro plasma stability of human anti-CD70 antibody drug conjugate, MDX-1203

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC MDX-1203 is a human anti-CD70 antibody conjugated to a pro-drug containing DNA alkylating cytotoxic Drug A. Drug A is composed of the anti-tumor compound Drug B, containing an ester linked protecting group, and a maleimide containing cleavable peptide linker designed to facilitate conjugation to the antibody. Mechanism of action of ADCs involve antibody mediated tumor specific delivery, cellular uptake, and intracellular pro-drug release and activation. Understanding the stability of the peptide linker as well as the pro-drug in various plasma matrices is critical for the development of successful ADC therapeutic. We have generated mouse monoclonal antibodies that specifically recognize either the intact Drug A or Drug A without the protecting group. Using these antibodies, an ELISA method was developed to study in vitro plasma stability of pro-drug in MDX-1203. Our results indicate that the plasma stability of ester linked protecting group varies significantly depending on the animal species probably due to variation in the level of plasma esterase activity. In order to determine the peptide linker stability, a radiolabeled MDX-1203 with 14C radiolabel on Drug A was made. A Thin Layer Chromatography method was developed to determine the in vitro plasma stability of the peptide linker in different plasma matrices. Our results indicate that the peptide linker in MDX-1203 is stable at 37o C up to 6 days. Based on these experimental data, we propose a model for stability of the peptide linker as well as the pro-drug in MDX-1203 in various plasma matrices. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2587.

Abstract 2575: Activation of antibody drug conjugate MDX-1203 by human carboxylesterase

MDX-1203 is a human anti-CD70 antibody conjugated to a DNA alkylating cytotoxic Drug A. Drug A is an enzyme-activated prodrug that requires cleavage of its peptide linker and removal of an ester-linked protecting group for cytotoxic activity. Upon internalization of MDX-1203 in targeted cancer cells, peptide linker in drug A is cleaved by lysosomal enzymes to produce Drug B; which is further processed by intracellular enzymes to produce active drug. The purpose of this study was to determine if Drug B and n-acetyl-cysteine-conjugated Drug A (NAC-Drug A) are substrates of recombinant human carboxylesterase, and to determine kinetic parameters for the enzyme iso-forms rhCE1 and rhCE2. Furthermore, a comparison of rhCE1 and rhCE2 kinetics was made with CPT-11, a commercially available prodrug used to treat colon cancer that is preferentially activated by human CE2. Human carboxylesterase 1 and 2 with a C-terminal histidine tag were cloned and expressed in CHO cells. The enzymes were purified using immobilized metal ion affinity chromatography (IMAC), and their identities were confirmed by N-terminal protein sequencing and Western blot using an anti-his antibody. Enzyme reactions were carried out at 37°C by incubation with Drug A, NAC-Drug B, or CPT-11. The amount of enzymatically released drug product was determined by RP-HPLC. Drug B was determined to be a substrate for both rhCE1 and rhCE2; however, its K m was much lower with rhCE2 (1.5 uM) as compared to rhCE1 (53 uM), and the overall enzyme efficiency (V max /K m ) was approximately 50 fold higher for rhCE2 as compared to rhCE1. Similarly, Drug B and NAC-Drug A were both found to be better substrates for rhCE2 as compared to rhCE1. Furthermore, recombinant human CE2 cleaves ester group in NAC-Drug A, 20 fold more efficiently as compared to CPT-11. These results indicate that expression of carboxylesterase 2 in targeted cancer cells is probably important for intracellular activation of MDX-1203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2575.

Abstract 2446: Biodistribution of antibody drug conjugates by LC-MS

Introduction: A method to study biodistribution of adduct forming antibody drug conjugates (ADCs) by LC-MS Description: ADCs are increasingly used to deliver cytotoxic payloads to tumor cells. While the targeted approach has vastly improved the toxicity profiles of these drugs, it is still important to understand the bio-distribution and the enhanced tumor localization this approach offers. Radio-labeled methods are still the most reliable approach to study the bio-distribution, but may not be able to differentiate between the drug that is present in the tissue from those that have formed covalent adducts upon non-specific internalization. Here, we describe a mass-spectrometric methods to identify these covalent adducts these cytotoxic agents form and how such methods could be utilized to map the bio-distribution in xenograft models. Data: The antibody drug conjugate is highly localized in the tumor with a %ID/gm value of almost 40 within 24 hours of dosing and no non-specific accumulation in other organs observed. Conclusions: A simple and robust LC-MS based method has been developed to understand the bio-distribution of antibody-drug conjugates by detecting and quantifying the actual and final chemical state of the toxin in the targeted cell. This method could be used to evaluate the (1) PK of antibody-drug conjugates (2) make lead selections at advanced preclinical stages of antibody-drug development (3) understand the ADME properties of the chosen antibody-drug conjugate Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2446.

Abstract 2452: Efficacy and toxicity of an anti-CD19 antibody drug conjugate

CD19 is an attractive target for immunotherapy. The expression of CD19 is restricted to B cells and follicular dendritic cells. In B cells, expression is initiated at the point of B lineage commitment and continues through the pre B and mature B cell differentiation. Expression is lost during the terminal differentiation of B cells to plasma cells. CD19 expression continues in B-lineage cells that have undergone neoplastic transformation, making it a useful marker in diagnosis and treatment of B cell derived lymphomas and leukemias, including non-Hodgkin9s lymphoma (NHL), acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We have generated a fully human antibody to CD19 (MDX-1435) in transgenic mice for high binding affinity as well as the ability to rapidly internalize for drug delivery. The antibody was conjugated to a potent DNA alkylating agent related to Duocarmycin (MED-2460). This ADC (MDX-1206) shows anti-tumor activity in established Raji, Daudi and Ramos subcutaneous xenograft models at doses as low as 0.1 μmole/kg with no associated weight loss, or other overt toxicity. Efficacy was substantially superior to that of MDX-1435 and non-specific control antibody conjugates. In addition, MDX-1435 binds to cynomologus monkey CD19 with similar affinity and tissue distribution as in humans. The intravenous administration of two doses at 0.4 μmole/kg was well tolerated in cynomologus monkeys and did not result in overt clinical or histological signs of toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2452.