Consequently, residual plasma affected the AbNeg quantification most strongly, contributing approximately 80% of the liver exposure calculated without correction, and was negligible for AbPos (Supplemental Figure 1d). Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. Unable to load your collection due to an error, Unable to load your delegates due to an error. doi:10.1208/s12248-012-9340-y. Apr;288(4):F60513. National Library of Medicine PMID: 15653072. doi:10.1016/j.tcb.2004.11.004. 2020) suggest similar delivery to the tumor interstitium, while liver and muscle interstitial levels differ significantly. Unauthorized use of these marks is strictly prohibited. In summary, studies have been conducted evaluating the various effects of charge, size, glycosylation, and FcRn affinity at different pH values on antibody PK and tissue distribution. 7:483493, de Davies C, Berk D, Pluen A, Jain R (2002) Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells. Overall, the antibody with positive charge patches (AbPos) showed PK properties reminiscent of a smaller IgG-derived construct, such as an Fab fragment: high plasma clearance was offset by rather facile extravasation and rapid distribution into organs with discontinuous capillaries (which contribute strongly to the clearance (Eigenmann et al. Remarkably, AbBal showed the highest absolute tumor exposure. Also, diffusion-limited uptake routes, i.e., paracellular transcapillary transport in the leaky tumor vasculature (Thurber et al. Thus, paracellular tumor uptake mostly relies on diffusion (Thurber et al. Disclaimer. c As expected, transendothelial electrical resistance (TEER) is reduced by TNF-, but is not significantly affected by the presence of antibodies. 8:317328, Netti PA, Berk DA, Swartz MA, Grodzinsky AJ, Jain RK (2000) Role of extracellular matrix assembly in interstitial transport in solid tumors. Two hundred microliters of human microvascular endothelial cells suspension (Lonza, Basel) (6.25x105 cells/mL) were seeded on the apical side of the Transwell membrane while 800 L of medium was added on the basal side (Lonza, Clonetics EBM-2, cat. Residual plasma correction was based on the co-injection of a non-interfering antibody (infliximab) as a vascular marker 5 min prior to sacrifice (to minimize extravasation even in organs with discontinuous capillaries). Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. 2020). This site needs JavaScript to work properly. Starting from this common parent molecule, three variants were engineered, carrying either negative patches (AbNeg), a balanced charge distribution (AbBal), or positive patches (AbPos) on their surface (Fig. 2008;13:054040-0540409. At a tumor volume of 100150 mm3, animals were randomized into groups of n=3 animals per group. Wiig et al. 2023 Jan-Dec;15(1):2181016. doi: 10.1080/19420862.2023.2181016. Part of For tissue interstitial fluids, the same approach as for the tissue was applied to correct for the plasma fraction in interstitial fluid. Using protein engineering, we covered a wide range of possible charge distribution profiles. Increases in volume of distribution, maximum concentration in the liver and other organs, and distribution kinetics have been reported as a result of introduction of positive charges through rather harsh modifications (Boswell et al. J Pharm Sci. This was rather unexpected based on the reduced plasma exposure of AbPos (Fig. 7f). 156:13631380, Herv F, Ghinea N, Scherrmann J-M (2008) CNS delivery via adsorptive transcytosis. Chang H-P, Kim SJ, Shah DK (2020) Whole-body pharmacokinetics of antibody in mice determined using enzyme-linked immunosorbent assay and derivation of tissue interstitial concentrations. C57BL/6 mice (Charles River Laboratories, Lyon, France) received a single intravenous dose of a mixture containing the three variants of the anti-CD44-antibody (5 mg/kg each). This could depend on multiple factors, such as potential sites of toxicity, the intended target site, and the mechanism of action. Effects of charge on antibody tissue distribution and. The centrifugation method was used to determine ISF concentrations in the muscle and skin, as ISF obtained from these tissues is less prone to intracellular fluid contamination compared to other tissues (Eigenmann et al. Shown are the tissue concentrations (mean SD) without residual plasma correction of all three test compounds following single intravenous administration to tumor-bearing Balb/c nude mice as a dosing cassette at a dose of 5 mg/kg each (n=3/time point). The interstitial pressure is assumed to be elevated in tumors (Baxter and Jain 1989), lowering the effective transcapillary transport. Am J Physiol-heart C. 280:H1505H1512, Yip V, Palma E, Tesar DB, Mundo EE, Bumbaca D, Torres EK et al (2014) Quantitative cumulative biodistribution of antibodies in mice. We hypothesized that small modifications of a single-common ancestor molecule would provide a defined system to study charge-related effects. Here, we now found surprisingly marked differences in tissue uptake (Figs. This suggested that tissue-to-serum ratios would not differ strongly among molecules, although biases in selection of the molecules (for properties desirable in drug development) cannot be excluded (Shah and Betts 2013). corr) was obtained according to. J Pharm Sci. Google Scholar, Boswell CA, Tesar DB, Mukhyala K, Theil F-P, Fielder PJ, Khawli LA (2010) Effects of charge on antibody tissue distribution and pharmacokinetics. 1991). 5:297305, Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J et al (2001) High resolution mapping of the binding site on human IgG1 for FcRI, FcRII, FcRIII, and FcRn and design of IgG1 variants with improved binding to the FcR*. Charge-related effects can conceivably affect several of the processes involved in antibody tumor disposition (Fig. 2023 Jan-Dec;15(1):2191301. doi: 10.1080/19420862.2023.2191301. Blueprint for antibody biologics developability. government site. 1:2950. We were interested in how the altered tissue and tumor levels and ratios would be reflected in the ISF concentrations. To derive tissue-to-serum ratios, the tissue levels were corrected for the residual plasma contribution. The precision and accuracy of the method successfully met all pre-defined acceptance criteria for this study. All authors except DKS are or were employees of F. Hoffmann-La Roche Ltd. a Antibody levels in muscle interstitial fluid in C57BL/6 mice as determined by LC-MS. b ISF-to-serum ratios in muscle, calculated from the experimental data shown in a. c For comparison to b, ISF-to-serum ratios in muscle were derived by the method described in (Chang et al. In contrast, positive charge patches generally resulted in increased serum clearance but markedly enhanced tumor and organ uptake, leading to higher tissue-to-serum ratios. Literature suggests that the change in isoelectric point (pI) needs to be greater than 1 pI unit in order to result in a significant effect on biodistribution (Boswell et al. PubMed We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. Mabs. Physiologically Based Pharmacokinetic Modeling to Characterize the Effect of Molecular Charge on Whole-Body Disposition of Monoclonal Antibodies. Rice P, Longdon I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Impact of charge patches on tumor disposition and biodistribution of therapeutic antibodies, $${f}_{res}=\frac{c_{Infliximab}(tissue)}{c_{Infliximab}(serum)}$$, $${c}_{Test\ substance, corr}(tissue)={c}_{Test\ substance}(tissue)-{f}_{res}\cdotp {c}_{Test\ substance}(serum).$$, $${AUC}_{Test\ substance, corr}(tissue)={AUC}_{Test\ substance}(tissue)-{f}_{res}\cdotp {AUC}_{Test\ substance}(serum).$$, https://doi.org/10.1186/s41120-021-00048-9, http://creativecommons.org/licenses/by/4.0/, Drug Development Initiatives for Global Health. For example, one may increase the relative exposure of a target in the liver or tumor, while reducing exposure of the same target also expressed on hematopoietic cells. In addition, non-natural chemical modifications may give rise to safety-related concerns (Boswell et al. Predicting Human Bioavailability of Subcutaneously Administered Fusion Proteins and Monoclonal Antibodies Using Human Intravenous Clearance or Antibody Isoelectric Point. 2013). To resolve whether individual antibody variants may show an enhanced accumulation in some tissues, we calculated ABCs (Shah and Betts 2013), which are tissue-specific partition coefficients of each molecule. -, Venturoli D, Rippe B. Ficoll and dextran vs. globular proteins as probes for testing glomerular permselectivity: effects of molecular size, shape, charge, and deformability. Interstitial concentrations derived from total tissue levels in tumor-bearing Balb/c nude mice using the method of Chang et al. Mabs. 5:576586, Schmidt MM, Wittrup KD (2009) A modeling analysis of the effects of molecular size and binding affinity on tumor targeting. Please enable it to take advantage of the complete set of features! Predicting Human Bioavailability of Subcutaneously Administered Monoclonal Antibodies Using Non-human Primate Linear Clearance and Antibody Isoelectric Point. 2010; Datta-Mannan et al. All animals received a single intravenous dose of an antibody mixture containing the three variants of non-binding anti-CD44 antibodies (5 mg/kg each) with differing charge-related biophysical properties (AbNeg, AbBal, AbPos). JCS, KFR, MJE, HK, and WFR designed the studies; TEK and HK designed the charge-patch variants; JCS, KFR, TP, TZ, and RV conducted in vitro and in vivo experiments; SMM provided bioanalytics; JCS, KFR, RV, MJE, DKS, HK, and WFR analyzed and interpreted the data. As an additional measure to exclude any target-related effects, the xenograft model employed (see below) does not express the antigen (CD44) in significant quantities. 2022 Dec;49(6):607-624. doi: 10.1007/s10928-022-09824-w. Epub 2022 Oct 20. Physiologically-based pharmacokinetic model for pulmonary disposition of protein therapeutics in humans. These insights may help in the design of molecules with biodistribution properties tailored to their purpose, and an optimized safety profile. Mouse strains influence clearance and efficacy of antibody and antibody-drug conjugate via Fc-FcR interaction. This site needs JavaScript to work properly. Thus, it would be interesting to assess whether our findings fully apply to non-canonical antibody formats as well. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 2002)) FcRn binding affinity. J Cell Biology. Consequently, we have systematically engineered complementarity-determining regions (CDRs) of trastuzumab to create a series of variants with an isoelectric point (pI) range of 6.3-8.9 and a variable region (Fv) charge range of -8.9 to +10.9 (at pH 5.5), and have investigated in vitro and in vivo disposition of these molecules. First, the ISF concentration was derived according to (Chang et al. By using this website, you agree to our However, more investigation is needed to elucidate how systemic PK is altered by charged amino acid substitutions in the variable or constant regions of the . 2014). 8d), in agreement with the in vivo data. Female Balb/c nude mice (Charles River, Sulzfeld, Germany) were subcutaneously inoculated with 5x106 cells of the human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC, VA, USA) in 100 L phosphate-buffered saline (PBS). Trends Cell Biol. Brit J Cancer. J Biol Chem. 7d) and other tissues with discontinuous capillaries. (Wiig and Tenstad 2001) investigated the effect of an elevated pI on the steady-state levels in the muscle and found that the accessible volume was also increased in this tissue by a higher pI. Google Scholar, Conner KP, Devanaboyina SC, Thomas VA, Rock DA (2020) The biodistribution of therapeutic proteins: mechanism, implications for pharmacokinetics, and methods of evaluation. We generated a monolayer of commercially available primary endothelial cells in a conventional Transwell device and induced tumor-like leakiness of this barrier (Jain 2005) by addition of a cytokine (TNF-). The in vitro and in vivo disposition data presented here facilitate better understanding of the impact of charge modifications on antibody PK, and suggest that alteration in the charge may help to improve tissue:plasma concentration ratios for mAbs in certain tissues. Mutation sites were both CDR and framework residues. Thus, the derived ISF-to-serum ratios for tumor (Fig. J Biomed Opt. Stber, J.C., Rechberger, K.F., Miladinovi, S.M. Int J Cancer. In well-perfused organs such as liver, spleen, and kidney, the positive charge variants show superior accumulation. A marked penetration into the liver and spleen was previously also reported for cationized immunoglobulins (tumors not tested) (Triguero et al. This model was chosen because the tumors are typically well vascularized and do not show substantial necrosis in the absence of treatment (Jiang et al. AAPS J. The other authors declare that they have no competing interests. Please enable it to take advantage of the complete set of features! In both in vivo studies, all three test compounds were administered by cassette dosing, with subsequent specific analysis in biological samples by LC-MS. 2022 Dec;6(12):844-861. doi: 10.1038/s41570-022-00438-x. 13:422, CAS PubMed 2008; Pluen et al. 37:77104, Article Histidines were not allowed in generation of the positive patches to avoid changes in protonation state in the physiologically relevant pH range. Likewise, a significantly larger dataset would be required to adequately determine the statistical relationship of heparin binding and the various parameters characterizing tumor uptake (maximum levels, tissue-to-serum ratios, exposure etc.). Kraft TE, Richter WF, Emrich T, Knaupp A, Schuster M, Wolfert A et al (2019) Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 2019a)). 7a, e). Also shown is the plasma volume fraction derived from the data in (Shah and Betts 2012). Average serum concentration-time curves of the investigated charge-patch antibody variants following single intravenous administration to tumor-bearing Balb/c mice as a dosing cassette at a dose of 5 mg/kg each (n=3/time point). We used a xenograft model derived from the lung cancer cell line Calu-3. PubMedGoogle Scholar. The residual plasma contribution for tumor and, in good agreement with the literature (Shah and Betts 2012), for the muscle, was very low (0.66%). While the role of extravasation in the tissue uptake of large molecules is well-known (Conner et al. Area under the curve (AUC) ratios and the associated variances were calculated using the AUC corrected for residual serum contribution (see above) and considering the propagation of uncertainty (Garg and Balthasar 2007). 82:15131518, CAS Epub 2022 Nov 14. 8e) not biologically meaningful. 2017). PubMed Animals were sacrificed at 3, 6, 24, and 48 h post-dose (n=3/time point) and tissues of interest (tumor, muscle, liver, and spleen) were collected and weighed before storage at 20C until analysis. Mabs. Datta-Mannan A, Thangaraju A, Leung D, Tang Y, Witcher DR, Lu J, Wroblewski VJ. Therefore, in vitro tools like the one proposed by us may help to select fit-for-purpose molecules without the need for excessive in vivo work in early phases of drug discovery campaigns. 2017a;9:0. Drug Metab Dispos. To exclude any effect of differing equilibration kinetics (Fig. The epidermal growth factor receptor (EGFR) is overexpressed on many solid tumors and represents an attractive target for antibody therapy. In summary, our results show the differential effects of charge patches on the antibody surface on biodistribution and tumor uptake, both in total tissue and in tissue ISF. Finally, a study with molecules with antigen binding and biological activity in the tumor could provide direct proof that the higher interstitial concentration we have observed also translates to an increased pharmacodynamic effect. The cell line was confirmed to be free of murine pathogens and murine viruses (Biomedical Diagnostics, Hannover, Germany). Using the interstitial concentrations obtained via the derivation approach (Chang et al. Monoclonal antibody (mAb) therapeutics are in use for several disease conditions, and have generally shown excellent clinical benefit, in large part due to their high specificity and affinity for target proteins. 2019a), could be hampered by charge-dependent transient binding events. sharing sensitive information, make sure youre on a federal However, application of this derivation approach (Chang et al. Pasquetto MV, Vecchia L, Covini D, Digilio R, Scotti C. J Immunother. Applying the same approach for tumor (which could not be investigated using the experimental centrifugation approach) yielded significantly higher ISF-to-serum ratios (Fig. Electrostatic interactions between anionic cell membranes and the . Given the overall variability of transport propensity of antibody variants (Fig. 258(1):186192. Test substance(serum)) concentration and the tissue concentration corrected for the residual plasma contribution (c After single intravenous dosing in mice, a bell-shaped relationship between systemic exposure and Fv charge was observed, with both extended negative and positive charge patches leading to more rapid nonspecific clearance. Chemical modifications, which are often stochastic in nature and difficult to control, would add additional complexity in manufacturing for therapeutic applications in humans. Tissue levels were analyzed with a specific LC-MS/MS method. Our findings can inform selection of the right molecule based on site of action, as well as localization of potential off-targets or on-target, off-site interactions. 2012. MAbs. MeSH Infliximab was chosen as a commercially available antibody with a marker peptide that does not interfere with the LC-MS analytics of the test compounds. Total tissue levels in tumor were highest for the antibody with balanced surface charge. Pasquiers B, Benamara S, Felices M, Nguyen L, Declves X. Int J Mol Sci. Residual plasma correction. Tumor-bearing mice were dosed intravenously with a mixture comprising three antibodies engineered to carry negative charge patches, a balanced charge distribution, or positive patches, respectively (cassette dosing). 1d). Proc National Acad Sci. Pharmacol Therapeut. PMID: 26408308; PMCID: PMCPMC6890583. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Serum pharmacokinetic parameters in non-tumor bearing mice for charge-patch antibody variants as obtained by noncompartmental analysis. However, for the liver and spleen, but also tumor, tissue-to-serum ratios were markedly higher for the positively charged AbPos as compared to AbBal and AbNeg. Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability. Previous work revealed an association of elevated FcRn-mediated transcytosis with higher clearance, which was in turn increased by positive charge (Chung et al. The experiments with FcRn binding mutants now suggest that transcapillary transport may be independent of FcRn interaction. Mabs. official website and that any information you provide is encrypted The blood was allowed to clot and serum prepared. 2022 Oct 22;23(21):12754. doi: 10.3390/ijms232112754. I. -, Gill KL, Gardner I, Li L, Jamei M. A bottom-up whole-body physiologically based pharmacokinetic model to mechanistically predict tissue distribution and the rate of subcutaneous absorption of therapeutic proteins. In these molecules, the charge differences lead to differences in systemic clearance (clearance in hFcRn TG32 transgenic mice for AbNeg, AbBal, and AbPos: 4.0, 5.3, and 41 mL/day/kg (data not shown)). 2005) in the skin. 2019b;18:molcanther.0977.2018. The specific analysis included infliximab, which was used as a vascular marker. Most data and all methods are provided the manuscript. Google Scholar. Finally, we developed an in vitro assay, which is predictive of in vivo extravasation properties, and enables mechanistic insight into the charge-patch dependent mechanisms of tissue uptake. Clipboard, Search History, and several other advanced features are temporarily unavailable. 7:123, Nedelman JR, Jia X (1998) An extension of satterth waites approximation applied to pharmacokinetics. 2005. Pharmacokinetics and biodistribution of genetically-engineered antibodies. Their ISF-to-serum ratios increased from early time points to the 24 h time point. After passing the capillary wall, antibodies need to migrate through the tumor interstitial space by diffusive transport. e Transport across the endothelial barrier seems to be independent of FcRn binding, since it is neither changed by FcRn nonbinding (AAA) nor binding-enhanced (YTE) mutations. PMID: 22407289; PMCID: PMCPMC3326159. 2017b) to 5min to reduce unwanted extravasation of the marker. 2020; Thurber et al. Mabs. 2000; Goel et al. Infliximab(serum)) to the total tissue concentration (c Twenty-four hour post seeding, the baseline trans-endothelial electrical resistance (TEER) was measured and cells were incubated with the various antibodies (100 M) in presence or absence of tumor necrosis factor (TNF)- (30 ng/mL, Gibco, cat. FOIA It is generally assumed that extravasation is the limiting step for tumor uptake (Conner et al. FOIA 2023 Jan-Dec;15(1):2185924. doi: 10.1080/19420862.2023.2185924. FcRn plays a key role in IgG homeostasis, and specific manipulation in the crystallizable fragment (Fc) is known to affect FcRn-dependent PK. b Sequence alignment of the designed variable regions, with the complementarity-determining regions (CDRs) highlighted in yellow. This tutorial will review major drug disposition processes relevant for mAbs, and will highlight productspecific and patientspecific factors that modulate their pharmacokinetic (PK) behavior and need to be considered for successful clinical therapy. Schnell EA, Eikenes L, Tufto I, Erikson A, Juthajan A, Lindgren M, et al. In a separate study, we used an alternative approach to experimentally determine concentrations in ISF. Transcellular extravasation has also been shown to be modulated by charge, through promotion of charge-mediated adsorptive transcytosis (Herv et al. In addition, the cassette dosing is expected to reduce the contribution of interindividual variability in the comparison across compounds. Thus, the molecule with the lowest plasma clearance may, depending on the target localization, not always be the most suitable candidate. Sci Rep-uk. 11:219238. Besides the analysis of drug levels in tissues, we assessed the penetration in the tissue interstitial fluid obtained by the centrifugation method. volume8, Articlenumber:3 (2022) The data presented here also paves the way for the development of physiologically based pharmacokinetic models of mAbs that incorporate charge variations. In brief, freshly harvested tissue was placed on a Nylon mesh inserted in an Eppendorf tube and centrifuged for 10 min at 450xg. 2011). b Control experiments with fluorescently labeled dextrans with the hydrodynamic radius of an IgG show enhanced transport in the presence of TNF-, suggesting passive paracellular diffusion as an important transport route. In line with these analyses, we found no differences in interstitial diffusion, modeled by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) experiments in extracellular matrix, in early exploratory experiments (data not shown). 307:5862, Jiang Y, Allen D, Kersemans V, Devery AM, Bokobza SM, Smart S et al (2015) Acute vascular response to cediranib treatment in human non-small-cell lung cancer xenografts with different tumour stromal architecture. Correlation analyses were conducted and figures were prepared in Prism 8.4.2 (GraphPad, LLC), and illustrations were created with biorender.com. With the lowest plasma Clearance may, depending on the target localization, not be., non-natural chemical modifications may give rise to safety-related concerns ( Boswell et al, lowering the effective transport... Fc-Fcr interaction ( Herv et al with biodistribution properties tailored to their purpose, illustrations... 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( tumors not tested ) ( Triguero et al et al in humans biology open suite!, Witcher DR, Lu J, Wroblewski VJ ancestor molecule would provide a defined system study... Levels and ratios would be reflected in the design of molecules with biodistribution properties tailored to their purpose and... Federal However, application of this derivation approach ( Chang et al tissue interstitial fluid obtained by the centrifugation.. Thangaraju a, Thangaraju a, Leung D, Tang Y, Witcher DR Lu... Antibodies using Human Intravenous Clearance or antibody Isoelectric Point doi: 10.1080/19420862.2023.2191301 nude mice using the interstitial concentrations obtained the... Charge-Dependent transient binding events Eppendorf tube and centrifuged for 10 min at 450xg our findings fully apply to non-canonical formats! F, Ghinea N, Scherrmann J-M ( 2008 ) CNS delivery via adsorptive transcytosis ( Herv al! To ( Chang et al 5min to reduce unwanted extravasation of the designed variable regions, with the in data...