C. Andrew Boswell

Effects of charge on antibody tissue distribution and pharmacokinetics.

Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. Emphasis must also be placed, however, on fundamental physicochemical properties that define antibodies as complex macromolecules, including shape, size, hydrophobicity, and charge. Electrostatic interactions between anionic cell membranes and the predominantly positive surface charge of most antibodies can influence blood concentration and tissue disposition kinetics in a manner that is independent of antigen recognition. In this context, the deliberate modification of antibodies by chemical means has been exploited as a valuable preclinical research tool to investigate the relationship between net molecular charge and biological disposition. Findings from these exploratory investigations may be summarized as follows: (I) shifts in isoelectric point of approximately one pI unit or more can produce measurable changes in tissue distribution and kinetics, (II) increases in net positive charge generally result in increased tissue retention and increased blood clearance, and (III) decreases in net positive charge generally result in decreased tissue retention and increased whole body clearance. Understanding electrostatic interactions between antibodies and biological matrices holds relevance in biotechnology, especially with regard to the development of immunoconjugates. The guiding principles and knowledge gained from preclinical evaluation of chemically modified antibodies will be discussed and placed in the context of therapeutic antibodies that are currently marketed or under development, with a particular emphasis on pharmacokinetic and disposition properties.

Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats.

Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.

Effects of Anti-VEGF on Pharmacokinetics, Biodistribution, and Tumor Penetration of Trastuzumab in a Preclinical Breast Cancer Model

Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography–X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes. Mol Cancer Ther; 11(3); 752–62. ©2012 AACR.

Conjugation of Monoclonal Antibodies with TETA Using Activated Esters: Biological Comparison of 64Cu-TETA-1A3 with 64Cu-BAT-2IT-1A3

A simple method for conjugation of monoclonal antibodies (mAbs) with the chelating agent 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), has been developed using commercially available reagents. This method involved activation of a single carboxyl group of TETA with N-hydroxysulfosuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The resulting activated ester of TETA was reacted with the anti-colorectal carcinoma mAb 1A3 at molar ratios ranging from 10:1 to 100:1 to give immunoconjugates modified with an average of 0.4 to 2.0 functional chelators per antibody molecule. The TETA-1A3 conjugate was labeled with 64Cu at specific activities as high as 15.4 microCi/microgram, and the radiolabeled mAb exhibited high in vitro serum stability and minimal loss of immunoreactivity. The biodistribution of 64Cu-labeled TETA-1A3 in hamsters bearing GW39 human colon carcinoma xenografts was compared to that of 64Cu-BAT-2IT-1A3 (BAT = 6-(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11- tetraacetic acid; 2IT = 2-iminothiolane). Both conjugates showed high tumor uptake (6.60-9.05% injected dose/gram) from 24 to 48 h post-injection and generally similar blood clearance and non-target organ uptakes. Human absorbed dose estimates derived from the hamster biodistribution data showed the critical organs for both conjugates to be the large intestine and the red marrow. Our results suggest that the in vitro and in vivo performance characteristics of 64Cu-TETA-1A3 compare favorably with those of 64Cu-BAT-2IT-1A3 and that further evaluation of the diagnostic and therapeutic efficacy of 64Cu-TETA-1A3 is warranted.

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21.

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.

Enhanced Tumor Retention of a Radiohalogen Label for Site-Specific Modification of Antibodies

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Comparative physiology of mice and rats: radiometric measurement of vascular parameters in rodent tissues.

A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed.

Tissue Physiology of Cynomolgus Monkeys: Cross-Species Comparison and Implications for Translational Pharmacology

We previously performed a comparative assessment of tissue-level vascular physiological parameters in mice and rats, two of the most commonly utilized species in translational drug development. The present work extends this effort to non-human primates by measuring tissue- and organ-level vascular volumes (Vv), interstitial volumes (Vi), and blood flow rates (Q) in cynomolgus monkeys. These measurements were accomplished by red blood cell labeling, extracellular marker infusion, and rubidium chloride bolus distribution, respectively, the same methods used in previous rodent measurements. In addition, whole-body blood volumes (BV) were determined across species. The results demonstrate that Vv, Vi, and Q, measured using our methods scale approximately by body weight across mouse, rat, and monkey in the tissues considered here, where allometric analysis allowed extrapolation to human parameters. Significant differences were observed between the values determined in this study and those reported in the literature, including Vv in muscle, brain, and skin and Q in muscle, adipose, heart, thymus, and spleen. The impact of these differences for selected tissues was evaluated via sensitivity analysis using a physiologically based pharmacokinetic model. The blood-brain barrier in monkeys was shown to be more impervious to an infused radioactive tracer, indium-111-pentetate, than in mice or rats. The body weight-normalized total BV measured in monkey agreed well with previously measured value in rats but was lower than that in mice. These findings have important implications for the common practice of scaling physiological parameters from rodents to primates in translational pharmacology.

Vascular physiology and protein disposition in a preclinical model of neurodegeneration.

The development of clinically relevant preclinical models that mimic the hallmarks of neurodegenerative disease is an ongoing pursuit in early drug development. In particular, robust physiological characterization of central nervous system (CNS) disease models is necessary to predict drug delivery to target tissues and to correctly interpret pharmacodynamic responses to disease-modifying therapeutic candidates. Efficient drug delivery across the blood-CNS barrier is a particularly daunting task, prompting our strategy to evaluate the biodistribution of five distinct molecular probes in a well-characterized mouse model of neurodegeneration. A transgenic mouse model of amyotrophic lateral sclerosis was selected based on a phenotype resembling clinical symptoms, including loss of motor neurons from the spinal cord and paralysis in one or more limbs, due to expression of a G93A mutant form of human superoxide dismutase (SOD1). The tissue distributions of two proteins, albumin and a representative immunoglobulin G antibody, as well as two blood flow markers, the lipophilic blood flow marker Ceretec (i.e., (99m)Tc-HMPAO) and the polar ionic tracer, rubidium-86 chloride ((86)RbCl), were measured following intravenous injection in SOD1(G93A) and age-matched control mice. The radiopharmaceutical TechneScan PYP was also used to measure the distribution of (99m)Tc-labeled red blood cells as a blood pool marker. Both the antibody and (86)Rb were able to cross the blood-spinal cord barrier in SOD1(G93A) mice to a greater extent than in control mice. Although the biodistribution patterns of antibody, albumin, and RBCs were largely similar, notable differences were detected in muscle and skin. Moreover, vastly different biodistribution patterns were observed for a lipophilic and polar perfusion agent, with SOD1(G93A) mutation resulting in reduced renal filtration rates for the former but not the latter. Overall, the multiprobe strategy provided an opportunity to efficiently collect an abundance of physiological information, including the degree and regional extent of blood-CNS barrier permeability, in a preclinical model of neurodegeneration.