Search

Oral delivery of therapeutic peptides has been challenging due to multiple physiological factors and physicochemical properties of peptides. We report a systematic approach to identify formulation compositions combining a permeation enhancer and a peptidase inhibitor that minimize proteolytic degradation and increase absorption of a peptide across the small intestine.An acylated glucagon-like peptide-1/glucagon co-agonist peptide (4.5 kDa) was selected as a model peptide. Proteolytic stability of the peptide was investigated in rat and pig SIF. Effective PEs and multiple component formulations were identified in rats. Relative bioavailability of the peptide was determined in minipigs via intraduodenal administration (ID) of enteric capsules.The peptide degraded rapidly in the rat and pig SIF. Citric acid, SBTI, and SBTCI inhibited the enzymatic degradation. The peptide self-associated into trimers in solution, however, addition of PEs monomerized the peptide. C10 was the most effective PE among tested PEs (DPC, LC, rhamnolipid, C12-maltosides, and SNAC) to improve intestinal absorption of the peptide in the rat IJ-closed loop model. A combination of C10 and SBTI or SBTCI increased the peptide exposure 5-tenfold compared to the exposure with the PE alone in the rat IJ-cannulated model, and achieved 1.06 ± 0.76% bioavailability in minipigs relative to subcutaneous via ID administration using enteric capsules.We identified SBTI and C10 as an effective peptidase inhibitor and PE for intestinal absorption of the peptide. The combination of SBTI and C10 addressed the peptide physiochemical properties and provides a formulation strategy to achieve intestinal delivery of this peptide.

An integrated physiologically based modeling framework is presented for predicting pharmacokinetics and bioavailability of subcutaneously administered monoclonal antibodies in cynomolgus monkeys, based on in silico structure-derived metrics characterizing antibody size, overall charge, local charge, and hydrophobicity. The model accounts for antibody-specific differences in pinocytosis, transcapillary transport, local lymphatic uptake, and pre-systemic degradation at the subcutaneous injection site and reliably predicts the pharmacokinetics of five different wild-type mAbs and their Fc variants following intravenous and subcutaneous administration. Significant associations were found between subcutaneous injection site degradation rate and the antibody's local positive charge of its complementarity-determining region (R = 0.56, p = 0.0012), antibody pinocytosis rate and its overall positive charge (R = 0.59, p = 0.00063), and antibody paracellular transport and its overall charge together with hydrophobicity (R = 0.63, p = 0.00096). Based on these results, population simulations were performed to predict the relationship between bioavailability and antibody local positive charge. In addition, model simulations were conducted to calculate the relative contribution of absorption pathways (lymphatic and blood), pre-systemic degradation pathways (interstitial and lysosomal), and the influence of injection site lymph flow on antibody bioavailability and pharmacokinetics. The proposed physiologically based modeling framework integrates fundamental mechanisms governing antibody subcutaneous absorption and disposition, with structured-based physiochemical properties, to predict antibody bioavailability and pharmacokinetics in vivo.

A model-based framework is presented to predict monoclonal antibody (mAb) pharmacokinetics (PK) in humans based on

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly 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. This 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. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal 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 those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

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.

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 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.

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.

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 IgG1-based 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 selection of IgG subclass during ADC drug discovery when employing stochastic cysteine conjugation.

Follistatin 315 heparan sulfate-binding deficient mutant human IgG4 Fc fusion (FST-ΔHBS-Fc) is a follistatin (FST) based Fc fusion protein currently being developed as a novel therapy for several potential indications, including muscle wasting. Previous assessments of the pharmacokinetics and therapeutic activity of FST-ΔHBS-Fc have shown a close association of the exposure-response relationship. The current work builds upon these initial studies by investigating the glycosylation characteristics of FST-ΔHBS-Fc after recombinant expression and its impact on the pharmacokinetics in mice and Cynomolgus monkeys. The data presented indicate that FST-ΔHBS-Fc is heterogeneously glycosylated at the three putative sites in FST when recombinantly expressed in stably transfected Chinese hamster ovary cells. Such carbohydrate heterogeneity, especially with regards to sialic acid incorporation, directly results in sugar-dependent clearance in both mice and Cynomolgus monkeys. Examination of the pharmacokinetics of FST-ΔHBS-Fc molecules containing variable sialic acid content in asialoglycoprotein receptor 1 (ASPGR-1) knockout mice supports the receptor's role as part of the clearance mechanism of the molecules. Based on the evaluation of several variably sialylated lots of material in pharmacokinetic assessments, we define specifications for average sialic acid incorporation into FST-ΔHBS-Fc that result in limited sugar-mediated clearance. Taken together, these studies highlight the importance of establishing an early understanding of the glycosylation/pharmacokinetic relationships of FST-ΔHBS-Fc, which will provide a basis for future application toward optimal systemic drug delivery and dosing strategies.

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.

Activin A, a member of the transforming growth factor-β superfamily, provides pleiotropic regulation of fibrosis and inflammation. We aimed at determining whether selective inhibition of activin A would provide a regenerative benefit. The introduction of activin A into normal muscle increased the expression of inflammatory and muscle atrophy genes Tnf, Tnfrsf12a, Trim63, and Fbxo32 by 3.5-, 10-, 2-, and 4-fold, respectively. The data indicate a sensitive response of muscle to activin A. Two hours after cardiotoxin-induced muscle damage, local activin A protein expression increased by threefold to ninefold. Neutralization of activin A with a specific monoclonal antibody in this muscle injury model decreased the muscle protein levels of lymphotoxin α and Il17a by 32% and 42%, respectively. Muscle histopathological features showed that activin A antibody-treated mice displayed an increase in muscle degradation, with the concomitant 9.2-fold elevation in F4/80-positive cells 3 days after injury. At the same time, the number of Pax7/Myod1-positive cells also increased, indicative of potentiated muscle precursor activation. Ultimately, activin A inhibition resulted in rapid recovery of muscle contractile properties indicated by a restoration of maximum and specific force. In summary, selective inhibition of activin A with a monoclonal antibody in muscle injury leads to the early onset of tissue degradation and subsequent enhanced myogenesis, thereby accelerating muscle repair and functional recovery.

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.

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 conformations, as well as, the number of FcRnBPs fused to the Fab both influence the clearance and the extent of pharmacokinetic benefit. FcRnBP fusion protein kinetics were also affected by a combination of post-translation modifications and non-specific binding properties. The results in this report lay some foundation in fostering the advent of newer technologies toward successfully improving the pharmacokinetics of proteins, peptides, and mAb-derived domains. Additional work in the integration of a variety of factors including the intended site of action, tissue disposition, metabolism, toxicity and pharmacokinetic, and pharmacodynamics relationship of the intended therapeutic modality are key areas for advancement of these approaches.

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-α (TNFα)). The T250Q/M428L anti-TNFα variant displayed an ∼40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFα variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased ∼500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFα T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFα variant displayed improved pharmacokinetics, characterized by an ∼2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.

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.

The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application.

The specificity of chemokine-receptor interactions plays a central role in the regulation of leukocyte migration in inflammatory responses. Herein, we describe a soluble mimic of CC chemokine receptor 2 (CCR2), dubbed CROSS-N(2)E3(2), which incorporates the N-terminal region (N) and third extracellular loop (E3) elements of CCR2 displayed on the surface of a soluble protein scaffold. CROSS-N(2)E3(2) binds to the CCR2 ligand monocyte chemoattractant protein-1 (MCP-1) with a dissociation equilibrium constant of 1.1 +/- 0.1 microM but does not bind to the cognate chemokines of the receptor CCR3 (eotaxin-1, -2, and -3). Similarly, a soluble analogue of CCR3 (CROSS(5)-N(3)E3(3)) binds to eotaxin-1, -2, and -3 but not to MCP-1. Thus, these receptor analogues have the same specificity as the natural receptors. Using soluble proteins containing N and E3 elements from different receptors (CROSS-N(2)E3(3) and CROSS-N(3)E3(2)), we demonstrate that both receptor elements are required for optimal binding to the cognate chemokines. In addition, we report the binding affinities of all four CROSS proteins to a panel of two wild-type and six chimeric chemokines. These complementation studies indicate the regions of the chemokines that interact with each element of the receptors, allowing us to deduce the orientations of the receptor extracellular elements relative to the bound chemokines.

Monoclonal antibodies (mAbs) represent an important class of therapeutic modalities. To optimize their pharmaceutical properties, studies have focused on improving mAb pharmacokinetic/pharmacodynamic profiles by modulating their interactions with the neonatal Fc receptor (FcRn). The influence of both the chemical and physical properties of IgGs has been examined in the context of FcRn interactions. In this regard, a variety of FcRn binding assays and tools have been developed and used to characterize the interaction with IgGs. However, a predictive relationship between the FcRn binding interaction of IgGs in vitro and their pharmacokinetics in vivo broadly across mAbs remains elusive. Many studies have increasingly suggested that the interplay between the characteristics of the mAb and the nature of its target can influence disposition and elimination. Thus, it is becoming increasingly evident that along with FcRn interactions, consideration of the non-FcRn-based biologic processes active in mAb disposition should be integrated into mAb development and optimization. Herein, we describe how the pharmacokinetics of mAbs can be modulated through FcRn interactions and provide perspectives on interpreting the receptor binding parameters in relation to other mechanisms involved in antibody disposition to aid in guiding mAb development.

Follistatin (FST) is a member of the tissue growth factor β family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.

Human follistatin is a regulatory glycoprotein with widespread biological functions including anti-inflammatory activities, wound healing properties and muscle stimulating effects. The role of follistatin in a wide range of biological activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential disease modifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic and pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315. Our data indicates that the intrinsic PK/PD properties of native follistatin-315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Herein, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing follistatin-315 to a murine IgG1 Fc and removing the intrinsic heparan sulfate binding activity of follistatin. The engineered variant molecule had ~100- and ~1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native follistatin-315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy towards the development of a follistatin therapeutic with potential utility in patients affected with skeletal muscle diseases.

The pH-dependent binding of IgGs to the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. Enhancing interactions between Fc and FcRn via protein engineering has been successfully used as an approach for improving the pharmacokinetics of monoclonal antibodies (mAbs). Although the quantitative translatability of the in vitro FcRn affinity enhancement to an in vivo pharmacokinetic benefit has been supported by several studies, there are also published reports indicating a disconnect in this relation. The body of literature suggests there are likely additional biochemical and biophysical properties of the mAbs along with their FcRn affinity that influence the in vivo pharmacokinetics. Herein, we more broadly evaluate the in vitro Fc-FcRn interactions and biochemical properties of five humanized IgG4 antibodies each with two Fc variant sequences (T250Q/M428L and V308P) and their corresponding pharmacokinetics in cynomolgus monkeys. Our findings indicate that the FcRn affinity-pharmacokinetic relationship does not show a direct correlation either across different IgGs or between the two variant sequences within a platform. Other parameters that have been suggested to contribute to mAb pharmacokinetic properties, such as the pH-dependent dissociation of the FcRn-IgG complexes, mAb biophysical properties, and nonspecific/charge binding characteristics of the mAbs, also did not independently explain the differing pharmacokinetic behaviors. Our results suggest that there is likely not a single in vitro parameter that readily predicts in vivo pharmacokinetics, but that the relative contribution and interplay of several factors along with the FcRn binding affinity are important determinants of mAb pharmacokinetic properties.

Engineering monoclonal antibodies (mAbs) with improved binding to the neonatal Fc receptor (FcRn) is a strategy that can extend their in vivo half-life and slow their systemic clearance. Published reports have predominantly characterized the pharmacokinetics of mAbs after intravenous administration. Recently, studies in mice suggest FcRn may also play a role in affecting the subcutaneous bioavailability of mAbs. Herein, we examined whether five mAbs engineered with the T250Q/M428L Fc mutations that improved their FcRn interactions, and subsequently their in vivo pharmacokinetics after intravenous administration, had improved subcutaneous bioavailability compared with their wild-type counterparts in cynomolgus monkeys. Similar to the intravenous administration findings, the pharmacokinetic profiles of our variant mAbs after subcutaneous injection showed improved half-life or clearance. In contrast, a clear effect was not observed on the subcutaneous bioavailability. We expect that while FcRn may play a role in determining mAb subcutaneous bioavailability, multiple biopharmaceutical and physiological factors are likely to influence the success of engineering strategies aimed at targeting this pathway for improving bioavailability.

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-alpha (TNFalpha)). The T250Q/M428L anti-TNFalpha variant displayed an approximately 40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFalpha variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased approximately 500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFalpha T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFalpha variant displayed improved pharmacokinetics, characterized by an approximately 2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.

It is well established that the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. As such, modification of the interaction of IgG with FcRn has been the focus of protein-engineering strategies designed to generate therapeutic antibodies with improved pharmacokinetic properties. In the current work, we characterized differences in interaction of IgG between mouse and primate receptors using three humanized anti-tumor necrosis factor alpha antibodies with variant IgG(1) Fc regions. The wild-type and variant IgG showed a differential combination of improved affinity, modified dissociation kinetics, and altered pH-dependent complex dissociation when evaluated on the primate and murine receptors. The observed in vitro binding differences within and between species allowed us to more completely relate these parameters to their influence on the in vivo pharmacokinetics in mice and cynomolgus monkeys. The variant antibodies have different pharmacokinetic behavior in cynomolgus monkeys and mice, which appears to be related to the unique binding characteristics observed with the murine receptor. However, we did not observe a direct relationship between increased binding affinity to the receptor and improved pharmacokinetic properties for these molecules in either species. This work provides further insights into how the FcRn/IgG interaction may be modulated to develop monoclonal antibodies with improved therapeutic properties.

To identify the elements of two chemokines [monocyte chemoattractant protein-1 (MCP-1) and eotaxin] that control their differential recognition by their respective receptors (CCR2 and CCR3), we have studied the receptor interactions of MCP-1-eotaxin chimeras. Each receptor was found to exhibit a distinct binding preference for proteins containing the amino-terminal region of the cognate chemokine for that receptor. However, other elements dictating chemokine preference were different for the two receptors. In some cases, the influence of replacing a particular region was dependent on the identities of neighboring regions, indicating a complex network of cooperative and/or compensating interactions.