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Papers by Amita Datta-Mannan

Monoclonal Antibodies: Past, Present and Future

Handbook of experimental pharmacology, 2019

Monoclonal antibodies (mAbs) are immunoglobulins designed to target a specific epitope on an anti... more Monoclonal antibodies (mAbs) are immunoglobulins designed to target a specific epitope on an antigen. Immunoglobulins of identical amino-acid sequence were originally produced by hybridomas grown in culture and, subsequently, by recombinant DNA technology using mammalian cell expression systems. The antigen-binding region of the mAb is formed by the variable domains of the heavy and light chains and contains the complementarity-determining region that imparts the high specificity for the target antigen. The pharmacokinetics of mAbs involves target-mediated and non-target-related factors that influence their disposition.Preclinical safety evaluation of mAbs differs substantially from that of small molecular (chemical) entities. Immunogenicity of mAbs has implications for their pharmacokinetics and safety. Early studies of mAbs in humans require careful consideration of the most suitable study population, route/s of administration, starting dose, study design and the potential difference in pharmacokinetics in healthy subjects compared to patients expressing the target antigen.Of the ever-increasing diversity of therapeutic indications for mAbs, we have concentrated on two that have proved dramatically successful. The contribution that mAbs have made to the treatment of inflammatory conditions, in particular arthritides and inflammatory bowel disease, has been nothing short of revolutionary. Their benefit has also been striking in the treatment of solid tumours and, most recently, as immunotherapy for a wide variety of cancers. Finally, we speculate on the future with various new approaches to the development of therapeutic antibodies.

* Background And Purpose: Many oncology ADCs have failed to demonstrate efficacy in clinic becaus... more * Background And Purpose: Many oncology ADCs have failed to demonstrate efficacy in clinic because of dose-limiting toxicity. We developed an approach that harnesses ADC affinity to broaden the therapeutic index (TI). * Experimental Approach: Two anti-mesenchymal-epithelial transition factor (MET) monoclonal antibodies (mAbs) with high affinity (HAV) or low affinity (LAV) were studied. IVM studies were conducted in rats to visualize and identify the cellular disposition of the mAbs within the liver and kidney. A rat HT-29 xenograft model was used to evaluate the in vivo anti-tumor efficacy of the two mAbs conjugated to monomethyl auristatin E (MMAE). The pharmacokinetics (PK) and toxicity profiles were also evaluated in rats. Single photon emission computed tomography (SPECT/CT) studies revealed the ADC tissue biodistribution, while mass spectroscopy measured free payload concentration in different organs. * Key Results: The estimated TI for LAV-ADC was at least 3 times greater than the HAV-ADC. The LAV-and HAV-ADCs showed similar levels of anti-tumor activity, while the 111In-DTPA studies showed simlar amounts of the ADCs in HT29 tumors. Although the LAV-ADC has slower blood clearance, higher liver toxicity was observed with HAV-ADC. While the SPECT/CT 111In-and 124I-DTPA findings showed HAV-ADC has higher accumulation and rapid clearance in normal tissues, the IVM studies confirmed HAV mAb accumulates within hepatic sinusoidal endothelial cells while the LAV mAb does not. * Conclusion And Implications: Lowering the MET binding affinity provides a larger TI for MET-ADC. Decreasing the affinity of the ADC reduces the TMDD to MET expressed in normal tissues while maintaining uptake/delivery to the tumor.

Clinical Pharmacology Applications of Real‐World Data and Real‐World Evidence in Drug Development and Approval–An Industry Perspective

Clinical Pharmacology & Therapeutics

Drug Metabolism and Disposition

Number of text pages: 26 Number of tables: 2 Number of figures: 6 (+ 5 supplementary figures) Num... more Number of text pages: 26 Number of tables: 2 Number of figures: 6 (+ 5 supplementary figures) Number of references: 45

Monoclonal Antibody Pharmacokinetics in Cynomolgus Monkeys Following Subcutaneous Administration: Physiologically Based Model Predictions from Physiochemical Properties

The AAPS Journal

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An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent dru... more An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs.

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Identification of a Multi-Component Formulation for Intestinal Delivery of a GLP-1/Glucagon Co-agonist Peptide

Pharmaceutical Research

Physiologically Based Modeling to Predict Monoclonal Antibody Pharmacokinetics in Humans from in vitro Physiochemical Properties

mAbs

Influence of FcRn binding properties on the gastrointestinal absorption and exposure profile of Fc molecules

Bioorganic & Medicinal Chemistry, 2021

The neonatal Fc receptor (FcRn) represents a transport system with the potential to facilitate ab... more The neonatal Fc receptor (FcRn) represents a transport system with the potential to facilitate absorption of biologics across the gastrointestinal barrier. How biologics interact with FcRn to enable their gastrointestinal absorption, and how these interactions might be optimized in a biological therapeutic are not well understood. Thus, we studied the absorption of Fc molecules from the intestine using three IgG4-derived Fc variants with different, pH-dependent FcRn binding and release profiles. Using several different intestinal models, we consistently observed that FcRn binding affinity correlated with transcytosis. Our findings support targeting FcRn to enable intestinal absorption of biologics and highlight additional strategic considerations for future work.

equilibrium dissociation constant; ELISA, enzyme linked immunosorbent assay; pH 50 , pH at which ... more equilibrium dissociation constant; ELISA, enzyme linked immunosorbent assay; pH 50 , pH at which 50% of the FcRn-antibody complexes dissociate; HRP, horseradish peroxidase; IV, intravenous; LLOQ, lower limit of quantitation; pI, isoelectric point; cIEF, capillary isoelectric focusing; T m , thermal stability; DSC, differential scanning calorimetry; AUC 0-∞ , area under the plasma concentration curve from zero to infinity; CL, clearance; C max , maximal observed plasma concentration; t 1/2 , elimination half-life; k off , dissociation rate This article has not been copyedited and formatted. The final version may differ from this version.

Antibodies, 2021

Bispecific antibodies (BsAb) that engage multiple pathways are a promising therapeutic strategy t... more Bispecific antibodies (BsAb) that engage multiple pathways are a promising therapeutic strategy to improve and prolong the efficacy of biologics in complex diseases. In the early stages of discovery, BsAbs often exhibit a broad range of pharmacokinetic (PK) behavior. Optimization of the neonatal Fc receptor (FcRn) interactions and removal of undesirable physiochemical properties have been used to improve the ‘pharmacokinetic developability’ for various monoclonal antibody (mAb) therapeutics, yet there is a sparsity of such information for BsAbs. The present work evaluated the influence of FcRn interactions and inherent physiochemical properties on the PK of two related single chain variable fragment (scFv)-based BsAbs. Despite their close relation, the two BsAbs exhibit disparate PK in cynomolgus monkeys with BsAb-1 having an aberrant clearance of ~2 mL/h/kg and BsAb-2 displaying a an ~10-fold slower clearance (~0.2 mL/h/kg). Evaluation of the physiochemical characteristics of the m...

mAbs, 2020

Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) admin... more Many therapeutic monoclonal antibodies (mAbs) were initially developed for intravenous (IV) administration. As a means to improve mAb drug-ability and the patient experience, subcutaneous (SC) administration is an increasingly important delivery route for mAbs. Unlike IV administration, bioavailability limitations for antibodies have been reported following SC injection and can dictate whether a mAb is administered via this parenteral route. The SC bioavailability of antibodies has been difficult to predict, and it can be variable and partial, with values ranging from ~50% to 100%. The mechanisms leading to the incomplete bioavailability of some mAbs relative to others are not well understood. There are some limited data that suggest the physiochemical properties inherent to a mAb can contribute to its SC absorption, bioavailability, and in vivo fate. In this study, we evaluated the integrated influence of multiple mAb physiochemical factors on the SC absorption and bioavailability of six humanized mAbs in both rats and cynomolgus monkeys. We demonstrate the physiochemical properties of mAbs are critical to their rate and extent of SC absorption. The combination of high positive charge and hydrophobic interaction significantly reduced the rate of the evaluated mAb's SC absorption and bioavailability. Reduction or balancing of both these attributes via re-engineering the mAbs restored desirable properties of the molecules assessed. This included reduced association with SC tissue, improvements in mAb absorption from the SC space and overall SC bioavailability. Our findings point to the importance of evaluating the relative balance between various physiochemical factors, including charge, hydrophobicity, and stability, to improve the SC drug-ability of mAbs for selecting or engineering mAbs with enhanced in vivo absorption and bioavailability following SC administration.

Antibodies, 2020

Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 ye... more Monoclonal antibodies have evolved from research tools to powerful therapeutics in the past 30 years. Clinical success rates of antibodies have exceeded expectations, resulting in heavy investment in biologics discovery and development in addition to traditional small molecules across the industry. However, protein therapeutics cannot drug targets intracellularly and are limited to soluble and cell-surface antigens. Tremendous strides have been made in antibody discovery, protein engineering, formulation, and delivery devices. These advances continue to push the boundaries of biologics to enable antibody conjugates to take advantage of the target specificity and long half-life from an antibody, while delivering highly potent small molecule drugs. While the “magic bullet” concept produced the first wave of antibody conjugates, these entities were met with limited clinical success. This review summarizes the advances and challenges in the field to date with emphasis on antibody conjug...

Abstract 353: A novel molecule with profound tumor killing activity

Experimental and Molecular Therapeutics, 2019

LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumo... more LY3343544: A novel MET antibody drug conjugate that shows profound pre-clinical in vivo anti-tumor activities, irrespective of MET pathway dependence MET is over-expressed in many types of human tumors. Due to the heterogeneity of human tumors, MET antibodies or small molecule inhibitors have benefited only small subsets of patients with tumors driven by signaling through the c-Met pathway. The patient selection strategies to identify those tumors with MET activation dependence are helpful in predicting sensitivity to many of these inhibitors. It was reported previously that Lilly’s MET antibody, emibetuzumab, showed clinical activity in selective NSCLC patients with high MET IHC staining (90% to 100% 3+ positive) when it was combined with erlotinib in Phase II clinical Trials. In searching for a better treatment for patients carrying the MET overexpression tumors regardless other co-existing mutations, we developed LY3343544, a novel antibody drug conjugate (ADC) molecule that consists of emibetuzumab conjugated with the potent microtubule inhibitor MMAE using a unique lysine conjugation approach. Upon binding to MET, LY3343544 is internalized via receptor-mediated endocytosis. LY3343544 maintains the similar binding and internalization activities to the cell surface MET as compared to emibetuzumab in the competitive cell binding assay and the internalization assay. We reported here that LY3343544 showed profound anti-tumor activity in a preclinical mouse models, and overcome intrinsic resistance mechanisms including KRAS, BRAF, PI3K and TP53 mutations. LY3343544 kills tumor cells expressing a wide range of MET levels on the cell surface and is capable of killing a variety of MET-overexpressing tumor cells including pancreatic, cholanglocarcinoma, colorectal, NSCLC, gastric, head and neck tumor cells in vitro. In contrast, LY3343544 does not kill human normal endothelial cells and normal epithelial cells, no activity on human peripheral blood mononuclear cells with or without activation as well as in cell-based assays. Moreover, LY3343544 is more stable in rodent PK studies than typical inter chain Cys VC-MMAE conjugates and showed tumor regressions in colorectal, NSCLC, gastric and pancreatic mouse xenograft models. Furthermore, LY3343544 shows profound tumor regression in >50% of PDAC PDX models (n=40): 20% complete response (CR); 22.5% partial response (PR); and 17.5% stable disease (SD); overall disease control rate (DCR) is 60%. In addition, LY3343544 shows tumor growth inhibition in cholangiocarcinoma PDX model that is resistant to emibetuzumab. In summary, LY3343544 is highly potent in killing a variety of tumor cells in cell-based killing assays. It demonstrated good stability in vivo and profound anti-tumor efficacy in multiple mouse xenograft models and patient-derived xenograft models thus is a promising agent to treat many types of cancers. Citation Format: Ling Liu, Aaron D. Wrobleski, Yin Yin, Wei Zeng, Xianming Chen, David J. Stokell, Sheng-bin Peng, Amita Datta-Mannan, Gregory P. Donoho, Philip W. Iversen, Philip Hipskind, Yiqing Feng. A novel molecule with profound tumor killing activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 353.

Biochemistry, 2019

There is a rapidly growing interest in the development of bi-or multi-specific antibody (BsAbs) b... more There is a rapidly growing interest in the development of bi-or multi-specific antibody (BsAbs) based biotherapeutics due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics, however, there is a paucity of such data for BsAbs. The present work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties, and in turn, how these relate to drug disposition. Biophysical characteristics of the molecules (charge, non-specific binding, FcRn and Fc receptor interactions, thermal stability, structure/dynamics and hydrophobic properties), indicated preferred orientations of ECD and IgG which supported better pharmacokinetic outcomes. In certain instances, where ECD-IgG configurations led to sub-optimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥ 2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with that of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAbs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs remains an opportunistic area for improving their in vivo kinetic properties.

Drug Metabolism and Disposition, 2019

Monoclonal antibodies (mAbs) and peptides are an important class of therapeutic modalities that h... more Monoclonal antibodies (mAbs) and peptides are an important class of therapeutic modalities that have brought improved health outcomes in areas with limited therapeutic optionality. Presently, there more than 90 mAb and peptide therapeutics on the United States market, with over 600 more in various clinical stages of development in a broad array of therapeutic areas, including diabetes, autoimmune disorders, oncology, neuroscience, and cardiovascular and infectious diseases. Notwithstanding this potential, there is high clinical rate of attrition, with approximately 10% reaching patients. A major contributor to the failure of the molecules is often times an incomplete or poor understanding of the pharmacokinetics (PK) and disposition profiles leading to limited or diminished efficacy. Increased and thorough characterization efforts directed at disseminating mechanisms influencing the PK and disposition of mAbs and peptides can aid in improving the design for their intended pharmacological activity, and thereby their clinical success. The PK and disposition factors for mAbs and peptides are broadly influenced by target-mediated drug disposition and nontarget-related clearance mechanisms related to the interplay between the relationship of the structure and physiochemical properties of mAbs and peptides with physiologic processes. This review focuses on nontarget-related factors influencing the disposition and PK of mAbs and peptides. Contemporary considerations around the increasing in silico approaches to identify nontarget-related molecule limitations and enhancing the druggability of mAbs and peptides, including parenteral and nonparenteral delivery strategies that are geared toward improving patient experience and compliance, are also discussed.

The AAPS Journal, 2018

Among the numerous antibody-drug conjugate (ADC) clinical candidates, one of the most prevalent t... more Among the numerous antibody-drug conjugate (ADC) clinical candidates, one of the most prevalent types utilizes the interchain cysteines in antibodies to conjugate auristatin via a maleimide-containing linker. In this class of ADCs, there are a paucity of systematic studies characterizing how IgG subclass influences the biophysical properties and in vivo pharmacokinetics of the ADC molecules. In the current investigation, we studied cysteine-conjugated ADCs using a model system consisting of human IgG1, IgG2, and IgG4 antibodies with the same variable region. Our findings identified some unforeseen differences among the three ADCs. Drug conjugation profiling by LC-MS revealed that 50% of inter heavy-light chain disulfide bonds are disrupted to conjugate drugs in IgG1 antibody while only 10% in IgG2 antibody and 20% in IgG4 antibody. The solution behavior of the ADCs was interrogated in concentrating experiments and diffusion interaction parameter measurements. We found that drug conjugation affected the solution property of the three antibodies differently, with the IgG2-based ADC having the most increased propensity to aggregate. Rat PK studies using a sensitive LC-MS-based bioanalytical method showed that the IgG1based ADC has poor peripheral linker-payload stability while the IgG2-and IgG4-based ADCs are stable. The conjugate stability of the IgG2-based ADC was further confirmed in a cynomolgus monkey PK study. Overall, the IgG2-based ADC exhibited the best PK/ conjugate stability but also the most deterioration in stability among the three ADCs. Our findings provide important information and present multifactorial considerations for the Electronic supplementary material The online version of this article (

Biotechnology journal, Jan 26, 2018

There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides... more There is a rapidly growing reinvigoration of the investigation of small proteins, cyclic peptides, and mAb derived domains as biotherapies. The drugability of these structures are challenged by fast peripheral clearance properties that can reduce their potential to be realized as medicines. Engineering strategies have been of limited value because mechanistically the half-life benefit is manifested by increasing the molecular weight and/or the hydrodyanimc radius which slows the molecule's renal elimination, but can result in the inherent loss of activity and target accessibility. The present work evaluated an alternative approach using smaller peptide sequences which bind to the neonatal Fc receptor (FcRn). Results revealed, small linear and cyclic FcRn binding peptides (FcRnBPs) fused to a combination of the N- and C-termini of a Fab can significantly improve the pharmacokinetics of the protein in cynomolgus monkeys relative to the parental Fab. The linear and cyclic conformat...