Targeting the Hemoglobin Scavenger receptor CD163 in Macrophages Highly Increases the Anti-inflammatory Potency of Dexamethasone
Authors: Jonas Heilskov Graversen, Pia Svendsen, Frederik Dagnæs-Hansen, Jakob Dal, Gabriele Anton, Anders Etzerodt, Mikkel D Petersen, Peter A Christensen, Holger J Møller, Søren K Moestrup
Journal: Molecular Therapy (2012)

Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery. Synthetic glucocorticoids are potent anti-inflammatory drugs but serious side effects such as bone mobilization, muscle mass loss, immunosuppression, and metabolic alterations make glucocorticoid therapy a difficult balance. The therapeutic anti-inflammatory effect of glucocorticoids relies largely on the suppressed release of tumor-necrosis factor-α and other cytokines by macrophages at the sites of inflammation. We have now developed a new biodegradable anti-CD163 antibody-drug conjugate that specifically targets the glucocorticoid, dexamethasone to the hemoglobin scavenger receptor CD163 in macrophages. The conjugate, that in average contains four dexamethasone molecules per antibody, exhibits retained high functional affinity for CD163. In vitro studies in rat macrophages and in vivo studies of Lewis rats showed a strong anti-inflammatory effect of the conjugate measured as reduced lipopolysaccharide-induced secretion of tumor-necrosis factor-α. The in vivo potency of conjugated dexamethasone was about 50-fold that of nonconjugated dexamethasone. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent dose of the conjugate had no such effect, measured as thymus lymphocytes apoptosis, body weight loss, and suppression of endogenous cortisol levels. In conclusion, the study shows antibody-drug conjugates as a future approach in anti-inflammatory macrophage-directed therapy. Furthermore, the data demonstrate CD163 as an excellent macrophage target for anti-inflammatory drug delivery. Synthetic glucocorticoids (GCs) such as dexamethasone and prednisone are widely used in the treatment of a range of severe inflammatory and autoimmune conditions.1Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids–new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2218) Google Scholar The synthetic GCs exert their effects via binding to the ubiquitous intracellular GC steroid receptor that in its ligand-binding conformation alters transcription of a large range of genes important for a diverse set of biological functions in metabolism, immunity, and bone/collagen formation.1Rhen T Cidlowski JA Antiinflammatory action of glucocorticoids–new mechanisms for old drugs.N Engl J Med. 2005; 353: 1711-1723Crossref PubMed Scopus (2218) Google Scholar The GC receptor is present in the cytoplasm of most types of cells, but the GC-induced gene expression profile depends on the cell type. The anti-inflammatory effect of GCs relates both to their effect on lymphocytes and on macrophages. 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In parallel to the development of antibody-drug conjugates (ADC), functioning as immunotoxins in cancer therapy,14Carter PJ Senter PD Antibody-drug conjugates for cancer therapy.Cancer J. 2008; 14: 154-169Crossref PubMed Scopus (341) Google Scholar we developed an anti-inflammatory ADC consisting dexamethasone linked to a monoclonal antibody against the macrophage-specific surface expressed endocytic receptor CD163. In humans, CD163 has been identified as the high affinity receptor for uptake of haptoglobin–hemoglobin complexes and a low affinity receptor for hemoglobin.15Kristiansen M Graversen JH Jacobsen C Sonne O Hoffman HJ Law SK et al.Identification of the haemoglobin scavenger receptor.Nature. 2001; 409: 198-201Crossref PubMed Scopus (1285) Google Scholar,16Schaer DJ Schaer CA Buehler PW Boykins RA Schoedon G Alayash AI et al.CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.Blood. 2006; 107: 373-380Crossref PubMed Scopus (228) Google Scholar CD163 is highly expressed in tissue macrophages in liver, spleen, and bone marrow, concordant with the high daily turnover of hemoglobin released into plasma due to physiological intravascular hemolysis (10–20% of total hemoglobin turnover). CD163 is also highly expressed on macrophages at sites of inflammation such as atherosclerotic lesions and inflamed joints in rheumatoid arthritis.17De Rycke L Baeten D Foell D Kruithof E Veys EM Roth J et al.Differential expression and response to anti-TNFalpha treatment of infiltrating versus resident tissue macrophage subsets in autoimmune arthritis.J Pathol. 2005; 206: 17-27Crossref PubMed Scopus (94) Google Scholar,18Komohara Y Hirahara J Horikawa T Kawamura K Kiyota E Sakashita N et al.AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.J Histochem Cytochem. 2006; 54: 763-771Crossref PubMed Scopus (151) Google Scholar,19Li W Xu LH Yuan XM Macrophage hemoglobin scavenger receptor and ferritin accumulation in human atherosclerotic lesions.Ann N Y Acad Sci. 2004; 1030: 196-201Crossref PubMed Scopus (16) Google Scholar CD163 is suggested to play an anti-inflammatory role by stimulating metabolism of the pro-inflammatory hemoglobin into its anti-inflammatory metabolites bilirubin and carbon monoxide.20Moestrup SK Møller HJ CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response.Ann Med. 2004; 36: 347-354Crossref PubMed Scopus (327) Google Scholar The high endocytic activity of CD163 further contributes to fast removal of hemoglobin20Moestrup SK Møller HJ CD163: a regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response.Ann Med. 2004; 36: 347-354Crossref PubMed Scopus (327) Google Scholar This study now demonstrates the first design, construction and characterization of an anti-inflammatory macrophage-targeting ADC generated by linking GC to an anti-CD163 mAb. The anti-inflammatory potential was studied by analysing the in vitro and in vivo efficacy in suppression of lipopolysaccharide (LPS)-induced inflammation. Figure 1 shows a schematic structure of the anti-CD163-dexamethasone conjugate synthesized by conjugating dexamethasone-hemisuccinate-NHS esters to the primary amino groups of the mouse anti-rat CD163 monoclonal antibody Ed-2 (anti-CD163), which binds specifically to macrophages in rat tissues.21Dijkstra CD Döpp EA Joling P Kraal G The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.Immunology. 1985; 54: 589-599PubMed Google Scholar,22Polfliet MM Fabriek BO Daniëls WP Dijkstra CD van den Berg TK The rat macrophage scavenger receptor CD163: expression, regulation and role in inflammatory mediator production.Immunobiology. 2006; 211: 419-425Crossref PubMed Scopus (155) Google Scholar In average, four dexamethasone molecules were conjugated per antibody with less than 1% remaining as free dexamethasone in the final preparations. Gel electrophoresis and size exclusion chromatography showed that the dexamethasone conjugation of anti-CD163 did not lead to the degradation or the formation of aggregates (Supplementary Figure S1 online). In a similar way, we synthesized anti-CD163 conjugated with the alternative GC analogues, prednisolone, and fluocinolone acetonide, which both have accessible hydroxyl-groups for hemisuccinate linkage (results not shown). Western blot analysis (Figure 2a) and flow cytometric binding analysis (Figure 2b) of Chinese hamster ovary (CHO) cells expressing rat recombinant CD163 (Figure 2a) showed maintained reactivity and specificity of anti-CD163 after dexamethasone conjugation. Accordingly, flow cytometry of rat spleen cells demonstrated specific binding of the antibody conjugate to CD172a and CD4 positive cells, i.e., monocytes/macrophages (Figure 2c). Analysis of transfected CHO cells expressing rat CD163 showed an efficient uptake of 125I-labeled anti-CD163, whereas no uptake was seen in nontransfected control CHO-cells (Figure 3). Degradation of the antibody measured as increase in the appearance of free 125I in the medium increased slightly after 2 hours compared with nontransfected cells. The uptake and cellular localization of anti-CD163-dexamethasone in the CHO cells as monitored by confocal microscopy showed uptake and intracellular colocalization of anti-CD163 and dexamethasone after 30 minutes (Figure 4a). After 2 hours, only partial colocalization of dexamethasone and the antibody was observed, thus indicating separation of the drug from the conjugate. Similar data were obtained for macrophage-like cells from rat spleen, whereas other nonmacrophage spleen cells did not exhibit measurable uptake of anti-CD163 or dexamethasone (Figure 4b).Figure 4Confocal fluorescence microscopy of the endocytotic uptake of anti-CD163-dexamethasone in CD163-expressing CHO transfectants and rat macrophages. A mixture of nontransfected and CD163-transfected CHO cells (a) or rat spleen cell suspensions (b) were incubated with anti-CD163-dexamethasone for 30 minutes and 2 hours before staining with anti-mouse IgG-Alexa Fluor 488 (green), anti-dexamethasone-PerCP (red), and Hoechst 33342 (light blue) nuclei staining. Colocation of anti-CD163 and dexamethasone is indicated by yellow color in bottom panels of a and b displays, marked by white arrow. Specificity was confirmed by lack of anti-CD163-dexamethasone binding in CD163 nonexpressing cells in the single displays. CHO, Chinese hamster ovary; TNF-α, tumor-necrosis factor-α.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The anti-inflammatory effect of anti-CD163-dexamethasone was examined in vitro by measuring LPS-mediated TNF-α production in rat macrophages incubated with free dexamethasone, anti-CD163-dexamethasone, control conjugate (IgG-dexamethasone), or nonconjugated anti-CD163 for 15 minutes (Figure 5) and then challenged with LPS after 18 hours. Anti-CD163-dexamethasone, control IgG-dexamethasone conjugates, and free dexamethasone (1 µg/ml) prevented LPS-mediated stimulation of rat macrophages, whereas anti-CD163 or vehicle alone had no effect on TNF-α secretion (data not shown). At dosing interval 10−3–10−6 mg/ml, the effect of anti-CD163-dexamethasone was significantly higher than that of free dexamethasone (P = 0.046) and IgG-dexamethasone (P = 0.028), and the effect of dexamethasone was higher than that of IgG-dexamethasone. The observed superior effect of anti-CD163 conjugated dexamethasone on the suppression of TNF-α secretion relative to free dexamethasone was only observed for shorter incubation times. When incubation was prolonged to 1 hour, the difference in effect between the two was abrogated (not shown). This probably reflects that passive diffusion of glucocorticoid in this system is slightly slower than the delivery via the CD163-mediated endocytosis. The anti-inflammatory effect of anti-CD163-dexamethasone in vivo was examined in rats injected with LPS that induces endotoxemia and acute inflammation.23Miksa M Das P Zhou M Wu R Dong W Ji Y et al.Pivotal role of the alpha(2A)-adrenoceptor in producing inflammation and organ injury in a rat model of sepsis.PLoS ONE. 2009; 4: e5504Crossref PubMed Scopus (62) Google Scholar,24Karpurapu M Wang X Deng J Park H Xiao L Sadikot RT et al.Functional PU.1 in macrophages has a pivotal role in NF-?B activation and neutrophilic lung inflammation during endotoxemia.Blood. 2011; 118: 5255-5266Crossref PubMed Scopus (60) Google Scholar Nineteen hours before the challenge with LPS, rats were i.v.-injected with dexamethasone, anti-CD163-dexamethasone, control-IgG-dexamethasone, or vehicle control. The inflammatory response to the LPS challenge was monitored by measuring TNF-α and IL-1 in the rat serum (Figure 6a). Dexamethasone was tested in two doses, a high dose (1 mg/kg) and a low dose (0.02 mg/kg). The high dose corresponds to the dose for maximal efficacy in other rat studies.25Li L Whiteman M Moore PK Dexamethasone inhibits lipopolysaccharide-induced hydrogen sulphide biosynthesis in intact cells and in an animal model of endotoxic shock.J Cell Mol Med. 2009; 13: 2684-2692Crossref PubMed Scopus (47) Google Scholar,26Hattori Y Murakami Y Atsuta H Minamino N Kangawa K Kasai K Glucocorticoid regulation of adrenomedullin in a rat model of endotoxic shock.Life Sci. 1998; 62: PL181-PL189PubMed Google Scholar The anti-CD163-dexamethasone conjugate was tested in two doses, where the dexamethasone content corresponded to the low dexamethasone content (0.02 mg/kg) and a fivefold lower dose (0.004 mg/kg). As compared with vehicle, a significantly lower TNF-α response was measured in animals treated with free high doses dexamethasone, whereas no effect was seen with the low dose. However, in the anti-CD163-dexamethasone form, the effect of the low dose (0.02 mg/kg) was comparable with the effect of the 1 mg/kg free dexamethasone dose. Similar data were obtained for serum IL-1, where the low doses of anti-CD163-dexamethasone almost completely abrogated the LPS-induced increase in IL-1. The inhibition of IL-1 induction by anti-CD163-dexamethasone was more efficient than that of free dexamethasone, even when this was at a 50-fold higher concentration (Figure 6b) (P < 0.02). Figure 7 shows the plasma disappearance of the therapeutic dose of anti-CD163-dexamethasone (2.4 mg protein/kg) in rats. Fifty percent of the conjugate disappeared from plasma in 20 minutes. The clearance curves of the anti-CD163 and dexamethasone moieties of the conjugate were virtually identical indicating a high stability of the conjugate in plasma (Figure 7). Furthermore, nonconjugated anti-CD163 showed a similar clearance profile as compared with anti-CD163-dexamethasone administered in the same doses (data not shown). However, the clearance of 125I-labeled anti-CD163 at a 50-fold lower dose (0.05 mg/kg) showed a half-life of 4 minutes due to uptake in mainly spleen and liver (Supplementary Figure S2 online). This indicates that the CD163 system is saturable. Comparison of the effect of the conjugate and high dose dexamethasone showed that two injections of 1 mg/kg free dexamethasone for acute therapy virtually erased cortisol from circulation after 2 days, whereas the equipotent dose (in terms of anti-inflammatory effect) of the conjugate had no significant effect on the cortisol levels (Figure 8a). Furthermore, we measured the effect on the weight of thymus, spleen, and whole body. The weight of the thymus after the administration of dexamethasone is a well-known parameter that reflects the corticosteroid-mediated apoptosis of lymphocytes, which are the dominating cells in the thymus.27Ben Rhouma K Sakly M Involution of rat thymus: characterization of cytoplasmic glucocorticoid receptors, evidence of glucocorticoid resistant dexamethasone receptor-positive cells.Arch Int Physiol Biochim Biophys. 1994; 102: 97-102PubMed Google Scholar,28Rungruang T Chaweeborisuit P Klosek SK Effect of malaria infection and dexamethasone on spleen morphology and histology.Southeast Asian J Trop Med Public Health. 2010; 41: 1290-1296PubMed Google Scholar,29Zhang Y Croft KD Mori TA Schyvens CG McKenzie KU Whitworth JA The antioxidant tempol prevents and partially reverses dexamethasone-induced hypertension in the rat.Am J Hypertens. 2004; 17: 260-265Crossref PubMed Scopus (69) Google Scholar Accordingly, two injections of 1 mg/kg free dexamethasone led to about a twofold reduction in thymus weight after 2 days as compared with the other groups (ANOVA test, P < 0.0001) (Figure 8b). No effect on thymus weight was observed for any of the other groups compared with vehicle-treated rats. Similar results were found for the spleen, which also undergoes a slight weight dexamethasone-induced reduction due to lymphocyte loss (Figure 8b) (ANOVA test, P < 0.0001). High dose weight dexamethasone injections (1 mg/kg) also reduce overall body (reduction of muscle, bone, and connective tissue) and a significant weight loss was seen compared with vehicle or dexamethasone conjugate injections (48 hours post first injection) (Figure 8d) (ANOVA, P = 0.0015). A prolonged study of systemic effects (Supplementary Figure S3 online) running for 9 days with four injections revealed a similar picture, but in this case with far more pronounced effect of the high dexamethasone dose on the weight of thymus (>75% reduction), spleen, and body. Endogenous cortisol was completely suppressed as in the short term study. The low concentrations of free dexamethasone exhibited significant reductions in the weight of thymus (P < 0.0001) and spleen (P < 0.002) as compared with vehicle. Conjugated dexamethasone at 0.02 mg/kg exhibited a small but significant reduction in thymus weight (P < 0.0001). Interestingly, low dose free dexamethasone has a significantly higher effect on thymus and spleen weight than the corresponding conjugated CD163 targeted dose (P = 0.013 and 0.039, respectively), thus indicating that not only the low concentration of dexamethasone, but also its targeting to macrophages reduces corticosteroid-mediated apoptosis of lymphocytes. The present study describes a rat anti-CD163 mAb-dexamethasone conjugate as a novel type of anti-inflammatory ADC. This anti-CD163-dexamethasone ADC has conserved binding affinity for CD163 and an in vivo potency about 50-fold higher than that of nonconjugated dexamethasone measured as reduction in LPS-induced TNF-α secretion. In contrast to a strong systemic effect of nonconjugated dexamethasone, the equipotent (in terms of effect on TNF-α secretion) dose of the ADC had no or low effect measured as thymus, spleen, and body weight reduction. According to the allometric conversion factor recommended by the Federal Drug Administration guidelines,30U.S. Department of Health and Human Services, F.a.D.A., Center for Drug Evaluation and Research 2005 Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers <http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm078932.pdf>Google Scholar the high dexamethasone dose used in this study corresponds to the upper end of dosing in human therapy and the low dose dexamethasone present in the conjugate represent the other end.30U.S. Department of Health and Human Services, F.a.D.A., Center for Drug Evaluation and Research 2005 Guidance for Industry, Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers <http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm078932.pdf>Google Scholar,31Mordenti J Man versus beast: pharmacokinetic scaling in mammals.J Pharm Sci. 1986; 75: 1028-1040Abstract Full Text PDF PubMed Scopus (350) Google Scholar As GSs represent the most potent class of anti-inflammatory drugs, it has been a long wish to develop GC analogues with a preferential anti-inflammatory effect compared with other effects that cause the severe adverse effects making prolonged GCs therapy hazardous. Whereas the regulation of GC potency and the reduction of unwanted mineralocorticoid effects of GCs have proved successful by changing side groups in the cyclopentanophenanthrene structure, it has not been possible to develop steroid variants with a specific anti-inflammatory effect avoiding the systemic GC effect. The reason is probably that the cytosolic GC receptor cannot mediate differentiated signals upon ligand binding. Our approach has therefore been to develop a low dose GC therapy with a specific high dose effect on macrophages including the macrophages at the sites of inflammation. The ADC approach is a well established method for directing cytostatic drugs to cancer cells and several drugs are in clinical development,32Teicher BA Antibody-drug conjugate targets.Curr Cancer Drug Targets. 2009; 9: 982-1004Crossref PubMed Scopus (125) Google Scholar but to our knowledge this is the first study of using the ADC approach for directing an anti-inflammatory drug to macrophages. 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One line of research has been to inject GC-loaded liposomes of various forms including negatively charged liposomes intended to target the macrophage scavenger receptor type 137Rensen PC Gras JC Lindfors EK van Dijk KW Jukema JW van Berkel TJ et al.Selective targeting of liposomes to macrophages using a ligand with high affinity for the macrophage scavenger receptor class A.Curr Drug Discov Technol. 2006; 3: 135-144Crossref PubMed Scopus (27) Google Scholar and mannosylated liposomes intended to target the mannose receptor.38Wijagkanalan W Higuchi Y Kawakami S Teshima M Sasaki H Hashida M Enhanced anti-inflammation of inhaled dexamethasone palmitate using mannosylated liposomes in an endotoxin-induced lung inflammation model.Mol Pharmacol. 2008; 74: 1183-1192Crossref PubMed Scopus (56) Google Scholar Also GC conjugated to mannosylated albumin has been studied.36Melgert BN Olinga P Van Der Laan JM Weert B Cho J Schuppan D et al.Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats.Hepatology. 2001; 34: 719-728Crossref PubMed Scopus (89) Google Scholar These studies have shown some effect in experimental animal models of arthritis, atherosclerosis and lung inflammation, although with an apparent much lower gain in efficacy compared with the present ADC approach. Whether this is due to difference in targeting efficacy, different target expression, or the use of different experimental conditions cannot be concluded at present. Our data showed a vesicular uptake in CD163-positive cells as expected (Figure 4). Due to the esterase-sensitive linkage and the general proteolytic milieu in the endocytic pathway, dexamethasone may be released in the lysosome and reach the cytosol by passive diffusion. This could suggest a longer response time but our in vitro efficacy study in rat macrophages indicated in fact a faster uptake of dexamethasone by CD163-mediated endocytosis (Figure 5) when it was conjugated to a high affinity anti-CD163 antibody. The overall gain in efficacy of the conjugated dexamethasone compared with free dexamethasone was far higher in the in vivo rodent system compared with the in vitro system with CD163-expressing rat macrophages in culture. Diffusion of the nonconjugated drug, but not the conjugated drug, into the entire pool of body cells (mostly CD163-negative) in the in vivo settings may explain this difference. The present data on a model of acute inflammation also opens exciting avenues for potential new treatment regimens using macrophage-targeted dexamethasone (or other GCs) in inflammatory diseases (e.g., rheumatoid arthritis and Mb. Crohn) where TNF-α has been validated as a target for biological drugs.6Ghezzi P Cerami A Tumor necrosis factor as a pharmacological target.Mol Biotechnol. 2005; 31: 239-244Crossref PubMed Scopus (42) Google Scholar It is tempting to speculate that macrophage-targeted dexamethasone may have an even more pronounced anti-inflammatory effect than anti-TNF-α biologicals because the macrophage-release of other pro-inflammatory cytokines also will be reduced as demonstrated for IL-1 in this