Cell Penetrating Peptides Conjugated to Anti-Carcinoembryonic Antigen "Catch-and-Release" Monoclonal Antibodies Alter Plasma and Tissue Pharmacokinetics in Colorectal Cancer Xenograft Mice.

Cell penetrating peptides conjugated to delivery vehicles, such as nanoparticles or antibodies, can enhance the cytosolic delivery of macromolecules. The present study examines the effects of conjugation to cell penetrating and endosomal escape peptides (i.e., TAT, GALA, and H6CM18) on the pharmacokinetics and distribution of an anti-carcinoembryonic antigen "catch-and-release" monoclonal antibody, 10H6, in a murine model of colorectal cancer. GALA and TAT were conjugated to 10H6 using SoluLINK technology that allowed the evaluation of peptide-to-antibody ratio by ultraviolet spectroscopy. H6CM18 was conjugated to either NHS or maleimide-modified 10H6 using an azide-modified valine-citrulline linker and copper-free click chemistry. Unmodified and peptide-conjugated 10H6 preparations were administered intravenously at 6.67 nmol/kg to mice-bearing MC38CEA+ tumors. Unconjugated 10H6 demonstrated a clearance of 19.9 ± 1.36 mL/day/kg, with an apparent volume of distribution of 62.4 ± 7.78 mL/kg. All antibody-peptide conjugates exhibited significantly decreased plasma and tissue exposure, increased plasma clearance, and increased distribution volume. Examination of tissue-to-plasma exposure ratios showed an enhanced selectivity of 10H6-TAT for the GI tract (+25%), kidney (+24%), liver (+38%), muscle (+3%), and spleen (+33%). 10H6-GALA and 10H6-H6CM18 conjugates demonstrated decreased exposure in all tissues, relative to unmodified 10H6. All conjugates demonstrated decreased tumor exposure and selectivity; however, differences in tumor selectivity between 10H6 and 10H6-H6CM18 (maleimide) were not statistically significant. Relationships between the predicted peptide conjugate isoelectric point (pI) and pharmacokinetic parameters were bell-shaped, where pI values around 6.8-7 exhibit the slowest plasma clearance and smallest distribution volume. The data and analyses presented in this work may guide future efforts to develop immunoconjugates with cell penetrating and endosomal escape peptides.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies.

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated KD of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (KD of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.

The impact of sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases.

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme. The N-glycan composition of 26BChE was compared to BChE with α-2,3 sialylation (23BChE) derived from wild-type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi-antennary di-sialylated glycans representing the most abundant glycoform. CD-1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.

Antibody Dependent Enhancement of Acinetobacter baumannii Infection in a Mouse Pneumonia Model.

Acinetobacter baumannii has become a pathogen of increasing medical importance because of the emergence of multidrug-resistant strains and the high rate of mortality of infected patients. Promising animal study results have been reported recently with active and passive immunization against A. baumannii virulence factors. In the present study, a monoclonal IgG3 antibody, 8E3, was developed with specificity for the K2 capsular polysaccharide of A. baumannii, and its therapeutic potential was assessed. 8E3 enhanced macrophage-mediated bactericidal activity against the A. baumannii clinical strain AB899. However, 8E3 treatment (passive immunization) of AB899-infected mice led to a substantial increase in mortality and to substantial increases in bacterial load in blood, lung, and in splenic samples. In vitro investigations showed a large binding capacity in the supernatant of bacterial cultures, suggesting that shed capsule components act as a binding sink for 8E3. Investigations of 8E3 pharmacokinetics in mice demonstrated that unbound concentrations of the antibody dropped below detection limits within 24 hours after a 200 mg/kg dose. However, total concentrations of antibody declined slowly, with an apparent terminal half-life (t 1/2) of 6.7-8.0 days, suggesting that the vast majority of 8E3 in blood is bound (e.g., with soluble capsule components in blood). We hypothesize that high concentrations of 8E3-capsule immune complexes act to inhibit bacterial clearance in vivo. To the best of our knowledge, this is the first demonstration of antibody-dependent enhancement of A. baumannii infection, and these observations highlight the complexity of antibody-based therapy for A. baumannii infections.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics.

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-µLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by µLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the µLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

"Catch-and-Release" Anti-Carcinoembryonic Antigen Monoclonal Antibody Leads to Greater Plasma and Tumor Exposure in a Mouse Model of Colorectal Cancer.

In this study, we examined the effects of target expression, neonatal Fc receptor (FcRn) expression in tumors, and pH-dependent target binding on the disposition of monoclonal antibodies (mAbs) in murine models of colorectal cancer. A panel of anti-carcinoembryonic antigen (CEA) mAbs was developed via standard hybridoma technology and then evaluated for pH-dependent CEA binding. Binding was assessed via immunoassay and radioligand binding assays. 10H6, a murine IgG1 mAb with high affinity for CEA at pH = 7.4 (KD = 12.6 ± 1.7 nM) and reduced affinity at pH = 6.0 (KD = 144.6 ± 21.8 nM), and T84.66, which exhibits pH-independent CEA binding (KD = 1.1 ± 0.11 and 1.4 ± 0.16 nM at pH 7.4 and 6.0), were selected for pharmacokinetic investigations. We evaluated pharmacokinetics after intravenous administration to control mice and to mice bearing tumors with (MC38CEA+, LS174T) and without (MC38CEA-) CEA expression and with or without expression of murine FcRn, at doses of 0.1, 1, and 10 mg/kg. 10H6 displayed linear pharmacokinetics in mice bearing MC38CEA+ or MC38CEA- tumors. T84.66 displayed linear pharmacokinetics in mice with MC38CEA- tumors but dose-dependent nonlinear pharmacokinetics in mice bearing MC38CEA+ In addition to the improved plasma pharmacokinetic profile (i.e., linear pharmacokinetics, longer terminal half-life), 10H6 exhibited improved exposure in MC38CEA+ tumors relative to T84.66. In mice bearing tumors with CEA expression, but lacking expression of murine FcRn (LS174T), 10H6 demonstrated nonlinear pharmacokinetics, with rapid clearance at low dose. These data are consistent with the hypothesis that pH-dependent CEA binding allows mAb dissociation from target in acidified endosomes, enabling FcRn-mediated protection from target-mediated elimination in mice bearing MC38CEA+ tumors.

Qualitative and quantitative characterization of protein biotherapeutics with liquid chromatography mass spectrometry.

In the last decade, the advancement of liquid chromatography mass spectrometry (LC/MS) techniques has enabled their broad application in protein characterization, both quantitatively and qualitatively. Owing to certain important merits of LC/MS techniques (e.g., high selectivity, flexibility, and rapid method development), LC/MS assays are often deemed as preferable alternatives to conventional methods (e.g., ligand-binding assays) for the analysis of protein biotherapeutics. At the discovery and development stages, LC/MS is generally employed for two purposes absolute quantification of protein biotherapeutics in biological samples and qualitative characterization of proteins. For absolute quantification of a target protein in bio-matrices, recent work has led to improvements in the efficiency of LC/MS method development, sample treatment, enrichment and digestion, and high-performance low-flow-LC separation. These advances have enhanced analytical sensitivity, specificity, and robustness. As to qualitative analysis, a range of techniques have been developed to characterize intramolecular disulfide bonds, glycosylation, charge variants, primary sequence heterogeneity, and the drug-to-antibody ratio of antibody drug conjugate (ADC), which has enabled a refined ability to assess product quality. In this review, we will focus on the discussion of technical challenges and strategies of LC/MS-based quantification and characterization of biotherapeutics, with the emphasis on the analysis of antibody-based biotherapeutics such as monoclonal antibodies (mAbs) and ADCs. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:734-754, 2017.

The Capsular Polysaccharide of Acinetobacter baumannii Is an Obstacle for Therapeutic Passive Immunization Strategies.

Acinetobacter baumannii has become an important concern for human health due to rapid development and wide spread of antimicrobial-resistant strains and high mortality associated with the infection. Passive immunizations with antisera targeting outer membrane proteins (OMPs) have shown encouraging results in protecting mice from A. baumannii infection, but monoclonal anti-OMP antibodies have not been developed, and their potential therapeutic properties have not been explored. The goal of this report is to evaluate the antibacterial activity of monoclonal antibodies (MAbs) targeting outer membrane protein A (OmpA) of A. baumannii Five anti-OmpA MAbs were developed using hybridoma technology and showed strong binding to strain ATCC 19606. However, low antibody binding was observed when they were tested against six clinical isolates, which included extensively drug-resistant strains. In contrast, high binding to an isogenic K1 capsule-negative mutant (AB307.30) was shown, suggesting that capsular polysaccharide mediated the inhibition of MAb binding to OmpA. Anti-OmpA MAbs increased the macrophage-mediated bactericidal activity of AB307.30 but failed to increase phagocytic killing of capsule-positive strains. Capsular polysaccharide was also protective against complement-mediated bactericidal activity in human ascites in the presence and absence of opsonization. Lastly, passive immunization with anti-OmpA MAbs did not confer protection against challenge with AB307-0294, the encapsulated parent strain of AB307.30, in a mouse sepsis infection model. These results reveal the important role of capsule polysaccharide in shielding OmpA and thereby inhibiting anti-OmpA MAb binding to clinical isolates. This property of capsule hindered the therapeutic utility of anti-OmpA MAbs, and it may apply to other conserved epitopes in A. baumannii.

Physiologically-based pharmacokinetic modeling to predict the clinical pharmacokinetics of monoclonal antibodies.

Accurate prediction of the clinical pharmacokinetics of new therapeutic entities facilitates decision making during drug discovery, and increases the probability of success for early clinical trials. Standard strategies employed for predicting the pharmacokinetics of small-molecule drugs (e.g., allometric scaling) are often not useful for predicting the disposition monoclonal antibodies (mAbs), as mAbs frequently demonstrate species-specific non-linear pharmacokinetics that is related to mAb-target binding (i.e., target-mediated drug disposition, TMDD). The saturable kinetics of TMDD are known to be influenced by a variety of factors, including the sites of target expression (which determines the accessibility of target to mAb), the extent of target expression, the rate of target turnover, and the fate of mAb-target complexes. In most cases, quantitative information on the determinants of TMDD is not available during early phases of drug discovery, and this has complicated attempts to employ mechanistic mathematical models to predict the clinical pharmacokinetics of mAbs. In this report, we introduce a simple strategy, employing physiologically-based modeling, to predict mAb disposition in humans. The approach employs estimates of inter-antibody variability in rate processes of extravasation in tissues and fluid-phase endocytosis, estimates for target concentrations in tissues derived through use of categorical immunohistochemical scores, and in vitro measures of the turnover of target and target-mAb complexes. Monte Carlo simulations were performed for four mAbs (cetuximab, figitumumab, dalotuzumab, trastuzumab) directed against three targets (epidermal growth factor receptor, insulin-like growth factor receptor 1, human epidermal growth factor receptor 2). The proposed modeling strategy was able to predict well the pharmacokinetics of cetuximab, dalotuzumab, and trastuzumab at a range of doses, but trended towards underprediction of figitumumab concentrations, particularly at high doses. The general agreement between model predictions and experimental observations suggests that PBPK modeling may be useful for the a priori prediction of the clinical pharmacokinetics of mAb therapeutics.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of gelonin in mouse plasma.

This article details the development and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of gelonin in mouse plasma. The ELISA was validated for intra- and inter-day variability and for accuracy over a standard curve range of 7.5-100 ng/mL. The assay was then applied to assess gelonin pharmacokinetics in mice. Results from the ELISA were compared to data obtained from a parallel study conducted with (125)Iodine-labeled gelonin, with quantification via gamma counting. The ELISA demonstrated good precision, as the percent coefficient of variation of quality control samples in intra-day and inter-day validation ranged from 5.4-9.3% and 2.9-7.3%, respectively. Sample recoveries ranged from 98.3-105% of nominal values. The ELISA method yielded lower plasma concentrations of gelonin than found from the less-specific gamma counting method. Consequently, pharmacokinetic analyses yielded significantly higher estimates for volume of distribution (106 ± 31 vs. 55.8 ± 13 mL/kg) and plasma clearance (34.7 ± 6.6 vs. 10.9 ± 2.1 mL/min/kg) for data determined by ELISA vs. by gamma counting.

Scale-up of a physiologically-based pharmacokinetic model to predict the disposition of monoclonal antibodies in monkeys.

Preclinical assessment of monoclonal antibody (mAb) disposition during drug development often includes investigations in non-human primate models. In many cases, mAb exhibit non-linear disposition that relates to mAb-target binding [i.e., target-mediated disposition (TMD)]. The goal of this work was to develop a physiologically-based pharmacokinetic (PBPK) model to predict non-linear mAb disposition in plasma and in tissues in monkeys. Physiological parameters for monkeys were collected from several sources, and plasma data for several mAbs associated with linear pharmacokinetics were digitized from prior literature reports. The digitized data displayed great variability; therefore, parameters describing inter-antibody variability in the rates of pinocytosis and convection were estimated. For prediction of the disposition of individual antibodies, we incorporated tissue concentrations of target proteins, where concentrations were estimated based on categorical immunohistochemistry scores, and with assumed localization of target within the interstitial space of each organ. Kinetics of target-mAb binding and target turnover, in the presence or absence of mAb, were implemented. The model was then employed to predict concentration versus time data, via Monte Carlo simulation, for two mAb that have been shown to exhibit TMD (2F8 and tocilizumab). Model predictions, performed a priori with no parameter fitting, were found to provide good prediction of dose-dependencies in plasma clearance, the areas under plasma concentration versu time curves, and the time-course of plasma concentration data. This PBPK model may find utility in predicting plasma and tissue concentration versus time data and, potentially, the time-course of receptor occupancy (i.e., mAb-target binding) to support the design and interpretation of preclinical pharmacokinetic-pharmacodynamic investigations in non-human primates.

Evaluation of near infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution.

The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of FDA-approved IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [(125)I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.

Investigation of the influence of nephropathy on monoclonal antibody disposition: a pharmacokinetic study in a mouse model of diabetic nephropathy.

PURPOSE: This study employed a mouse model to evaluate the effects of diabetic nephropathy on the pharmacokinetics of 8C2, a murine monoclonal antibody (mAb). METHODS: Streptozotocin (STZ) was administered to mice to induce diabetic nephropathy (125 mg/kg/day × 2). Mice were grouped (n = 8-10) based on time after STZ-treatment (control, 1, 2, 3, 4, or 6 weeks), and injected intravenously with 10 mg/kg 8C2. Blood samples were collected up to 7 days, and 8C2 plasma concentrations were determined via immunoassay. Inulin clearance and urinary albumin excretion rate (UAE) were determined to assess renal function. RESULTS: UAE, inulin clearance, and 8C2 clearance increased significantly following STZ. Comparing control and 6 week STZ-treatment groups, UAE and inulin clearance increased from 25.7 ± 3.3 to 99.3 ± 13.7 µg/day, and from 421 ± 31 to 584 ± 78 µl/min. 8C2 clearance increased from 121 ± 12.5 to 228 ± 61 µl/hr/kg (p < 0.01). 8C2 clearance was highly correlated with UAE (r(2): 0.731). Inclusion of UAE as a covariate in population modeling explained significant residual variability in 8C2 clearance. CONCLUSIONS: The clearance of 8C2 increased significantly in STZ-treated mice. Population pharmacokinetic modeling suggests that UAE has potential for use in predicting mAb clearance in subjects with diabetic nephropathy, possibly assisting in the individualization of mAb dosing.

Predicting the effects of 8C2, a monoclonal anti-topotecan antibody, on plasma and tissue disposition of topotecan.

We are investigating an inverse targeting strategy to reduce the dose limiting systemic toxicities resultant from intraperitoneal administration of topotecan, a model chemotherapeutic drug. This approach utilizes systemic co-administration of anti-topotecan antibodies to alter the plasma and tissue disposition kinetics of topotecan. To better predict the effects of 8C2, a high affinity anti-topotecan monoclonal antibody, on the pharmacokinetics of topotecan, two mathematical models have been developed and evaluated. Model 1 is a hybrid physiologically based pharmacokinetic (PBPK) model that was created by merging a PBPK model for topotecan with a simple two compartment model of 8C2 pharmacokinetics. Model 2 is a comprehensive PBPK model developed by merging a PBPK model for IgG with a PBPK model for topotecan. To help validate the simulation results from both the models, a tissue distribution experiment was conducted, in which topotecan and 8C2 were co-administered in mice. Experimental and simulated data were compared by calculating the median percent prediction error (%PE) for all tissues. For both models, the median %PE values for all the tissues were less than 100 %, indicating that the predicted values were, on average, less than twofold the observed plasma and tissue topotecan concentrations values. In general model 2 was found to be more predictive of the data set than model 1, as the overall median %PE value for model 2 (%PE = 63) was less than model 1 (%PE = 73).

Investigation of the role of FcγR and FcRn in mAb distribution to the brain.

To evaluate the role of Fc receptors (FcR) on IgG distribution to the brain, the disposition of 8C2, a murine monoclonal IgG1 antibody, was investigated after intravenous administration in FcRn α-chain knockout mice, FcγRIIb knockout mice, FcγRI/RIII knockout mice, and C57BL/6 control mice. (125)I-8C2 was co-administered with (51)Cr-labeled red blood cells to allow accurate assessment of residual blood content in brain samples. Blood and brain tissues were harvested from subgroups of three mice at several time-points up to 10 days, and radioactivity was counted. The blood and brain areas under 8C2 concentration vs time curves (AUCs) were calculated using the linear trapezoidal rule, and the associated standard deviations (SD) were assessed using a modified Bailer method. Concentration data were also analyzed with a semiphysiological population pharmacokinetic model. The brain/blood AUC ratios were comparable across all strains of mice (ratios ± SD): 0.00774 ± 0.000452, 0.00841 ± 0.000535, 0.00636 ± 0.000548, and 0.00917 ± 0.000478 for C57BL/6 control mice, FcγRI/RIII knockouts, FcγRIIb knockouts, and FcRn α-chain knockout mice (p > 0.05). Statistically significant improvement in model fitting of the data was shown with incorporation of a strain-specific parameter for antibody clearance for FcRn knockout mice; however, no significant improvements in model fitting were found for strain effects on any other parameter, including the brain uptake clearance or efflux clearances for 8C2. The predicted 8C2 brain efflux clearance was found to be ∼135-fold faster than the brain uptake clearance, consistent with the observed low ratio of brain-blood exposure. The experimental results and modeling results indicate that, in mice, FcRn and FcγR do not contribute to the "blood-brain barrier" that limits mAb uptake into the brain.

Second-generation minimal physiologically-based pharmacokinetic model for monoclonal antibodies.

Minimal physiologically-based pharmacokinetic (mPBPK) models provide a sensible modeling approach when fitting only plasma (or blood) data yielding physiologically-relevant PK parameters that may provide more practical value than parameters of mammillary models. We propose a second-generation mPBPK model specifically for monoclonal antibodies (mAb) by including (lumping) several essential components of mAb PK used in full PBPK models. These components include convection as the primary mechanism of antibody movement from plasma into tissues and return to plasma with interstitial fluid as the major extravascular distribution space. The model divides tissue spaces into two groups according to their vascular endothelial structure, leaky and tight, which consequently allows discernment of two types and general sites of distribution. This mPBPK model was applied to two mAbs in mice and ten mAbs with linear kinetics in humans. The model captured their plasma PK profiles well with predictions of concentrations in interstitial fluid for two types of tissues. Predictions of tissue concentrations for mAb 7E3 and 8C2 were consistent with actual measurements in mice, indicating the feasibility of this model in assessing extravascular distribution in the two categories of tissues. The vascular reflection coefficients (σ1) of tight tissues (V(tight)) ranged 0.883-0.987 and coefficients (σ2) for leaky tissues (V(leaky)) ranged 0.311 to 0.837. The plasma clearance (CL(p)) varied among the mAbs in humans from 0.0054 to 0.03 L/h. In addition, applying this model generates parameters for mAb transcapillary escape rates and assesses major sites of elimination. Four of ten mAbs exhibited better fitting statistics premised on elimination from interstitial fluid than from plasma. This approach allows comparisons of mAb PK when only plasma data are available, provides more realistic parameters and predictions than mammillary models, and may provide an intermediate step towards utilizing full PBPK models for mAbs.

Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability.

Anti-cancer antibody-drug conjugates (ADCs) aim to expand the therapeutic index of traditional chemotherapy by employing the targeting specificity of monoclonal antibodies (mAbs) to increase the efficiency of the delivery of potent cytotoxic agents to malignant cells. In the past three years, the number of ADCs approved by the Food and Drug Administration (FDA) has tripled. Although several ADCs have demonstrated sufficient efficacy and safety to warrant FDA approval, the clinical use of all ADCs leads to substantial toxicity in treated patients, and many ADCs have failed during clinical development due to their unacceptable toxicity profiles. Analysis of the clinical data has demonstrated that dose-limiting toxicities (DLTs) are often shared by different ADCs that deliver the same cytotoxic payload, independent of the antigen that is targeted and/or the type of cancer that is treated. DLTs are commonly associated with cells and tissues that do not express the targeted antigen (i.e., off-target toxicity), and often limit ADC dosage to levels below those required for optimal anti-cancer effects. In this manuscript, we review the fundamental mechanisms contributing to ADC toxicity, we summarize common ADC treatment-related adverse events, and we discuss several approaches to mitigating ADC toxicity.

Use of Payload Binding Selectivity Enhancers to Improve Therapeutic Index of Maytansinoid-Antibody-Drug Conjugates.

Systemic exposure to released cytotoxic payload contributes to the dose-limiting off-target toxicities of anticancer antibody-drug conjugates (ADC). In this work, we present an "inverse targeting" strategy to optimize the therapeutic selectivity of maytansinoid-conjugated ADCs. Several anti-maytansinoid sdAbs were generated via phage-display technology with binding IC50 values between 10 and 60 nmol/L. Co-incubation of DM4 with the anti-maytansinoid sdAbs shifted the IC50 value of DM4 up to 250-fold. Tolerability and efficacy of 7E7-DM4 ADC, an anti-CD123 DM4-conjugated ADC, were assessed in healthy and in tumor-bearing mice, with and without co-administration of an anti-DM4 sdAb. Co-administration with anti-DM4 sdAb reduced 7E7-DM4-induced weight loss, where the mean values of percentage weight loss at nadir for mice receiving ADC+saline and ADC+sdAb were 7.9% ± 3% and 3.8% ± 1.3% (P < 0.05). In tumor-bearing mice, co-administration of the anti-maytansinoid sdAb did not negatively affect the efficacy of 7E7-DM4 on tumor growth or survival following dosing of the ADC at 1 mg/kg (P = 0.49) or at 10 mg/kg (P = 0.9). Administration of 7E7-DM4 at 100 mg/kg led to dramatic weight loss, with 80% of treated mice succumbing to toxicity before the appearance of mortality relating to tumor growth in control mice. However, all mice receiving co-dosing of 100 mg/kg 7E7-DM4 with anti-DM4 sdAb were able to tolerate the treatment, which enabled reduction in tumor volume to undetectable levels and to dramatic improvements in survival. In summary, we have demonstrated the utility and feasibility of the application of anti-payload antibody fragments for inverse targeting to improve the selectivity and efficacy of anticancer ADC therapy.