Department of Microbiology, Immunology and Molecular Genetics and the Molecular Biology Institute, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095, USA, and 2Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo 1730015
Received on June 23, 2000; revised on August 14, 2000; accepted on August 16, 2000.
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Abstract |
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Key words: Fc carbohydrate/IgG catabolism
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Introduction |
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Antibodies are glycoproteins that have been extensively studied both because of their ability to specifically recognize foreign antigens and because of their diverse functional properties. Antibodies of different isotypes have different properties including different rates of in vivo clearance. Among the isotypes, IgG is relatively long-lived in vivo with a serum half-life in humans exceeding 21 days reported for some subclasses (Roitt, 1994). IgG is also distinguished by the fact that its rate of clearance is dependent on its serum concentration with increasing concentration associated with more rapid clearance (Brambell et al., 1964
).
The amino acid sequence of an antibody plays a role in determining its serum clearance. Different subclasses of IgG clear at different rates. In addition amino acid changes within the Fc region of the IgG can have profound effects on the rate of serum clearance (Kim, et al., 1994, 1999; Medesan, et al., 1997
; Zuckier et al., 1998
; Hornick et al., 2000
). In addition to amino acid sequence, the structure of the attached carbohydrate is thought to play a role in protein targeting and clearance (Mattes, 1987
; Varki, 1993
; Dwek, 1995
). We have shown that IgG1 antibodies whose Fc-associated carbohydrate has terminal mannose residues exhibit more rapid clearance than more fully glycosylated IgG1 antibodies (Wright and Morrison, 1994
). The present study is designed to further investigate the influence of the structure of the Fc-associated carbohydrate on the sites of clearance and organ targeting of IgG1.
The kinetics of protein clearance is traditionally measured by radiolabeling proteins with isotopes such as 125I and observing the rate of clearance of radioactivity either from the body of the experimental animal or from individual organs. However, when the protein is degraded the free label is rapidly lost from the tissue making it difficult or impossible to identify the site of catabolism of the protein.
To address these issues alternative labeling procedures using glycoconjugates have been developed. Glycoconjugates that are relatively large, hydrophilic, and resistant to lysosomal degradation remain for extended periods at the site of release from the protein and can serve as biologically inert tracers to identify in vivo sites of protein uptake and catabolism (Thorpe et al., 1993; Thorpe and Baynes, 1994
). Termed residualizing labels (R-labels), compounds such as dilactitol tyramine (DLT) have been developed which can be coupled to candidate proteins through mild reductive amination procedures that minimize damage to the protein and effects on the biological activity of the protein. In addition the residualizing labels can themselves be easily radioiodinated.
Several approaches have been used to alter the glycosylation state of IgG antibodies. These include inhibition of glycosylation by culturing cells in the presence of the drug tunicamycin an inhibitor of N-linked glycosylation (Leatherbarrow et al., 1985; Walker et al., 1989
), treatment of glycoproteins with specific glycosidases that remove the entire oligosaccharide or specific residues (Tsuchiya et al., 1989
; Boyd et al., 1995
) or site-directed mutagenesis to either remove the carbohydrate addition site (Tao and Morrison, 1989
) or residues within the CH2 region that contact the core oligosaccharide residues (Lund, et al., 1996
). However, a persisting concern with many of these approaches is that the treatment itself alters the conformation of the protein and hence influences its catabolism.
Production of immunoglobulin in Chinese hamster ovary (CHO) cells with defined glycosyltransferase mutations provides an alternative approach to studying the contribution of carbohydrate structure to antibody function. Compared to glycosidase digestion, this approach is advantageous in that: (1) homogeneous carbohydrate structures (at least in terms of what residues they lack) are produced without the potential complication of damage to the protein backbone through enzyme treatment; and (2) carbohydrate structures can be attached that are not easily produced by glycosidase treatment.
We have now expressed mousehuman chimeric IgG1 antibodies in wild-type Pro-5 CHO cells as well as in the glycosylation mutants Lec 1, Lec 2, and Lec 8. Lec 1 cells are deficient in N-acetylglucosaminyltransferase I and are expected to attach a truncated, Man5GlcNAc2 structure not normally seen on IgG. Lec 2 cells are defective in the transport of CMP-sialic acid and should synthesize a complex carbohydrate structure lacking sialic acid. Lec 8 cells fail to transport UDP-galactose, and should attach a complex carbohydrate structure lacking galactose to IgG1 (Stanley, 1984, 1987a,b). We have determined the structure of the carbohydrate attached to the purified antibodies used in this study to verify that mutant CHO cells do indeed attach carbohydrates of expected structure. We have now labeled these well-characterized proteins with a residualizing label and compared their organ targeting and clearance pathways. All proteins are catabolized throughout the body with the skin the major site of catabolism. In addition, the liver is a significant site of catabolism of IgG1 bearing the Lec 2 carbohydrate with terminal galactose and the Lec 1 carbohydrate with terminal mannose.
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Results |
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When oligosaccharides of IgG1-Lec 1 (peaks fi in Figure 1D) were digested with A.saitoi -mannosidase, which can cleave only the Man
1
2Man linkage (Yamashita et al., 1980
), peaks fi were all converted to the same position as component e, which showed the same mobility as an authentic Man
1
6(Man
1
3)Man
1
6(Man
1
3)Man-ß1
4Glc-NAcß1
4GlcNAc-2AB with the release of one, two, three, and four Man
1
2 residues (Figure 2D). The results indicated that oligosaccharides of IgG1-Lec 1 are a series of high mannose type containing one, two, three, and four Man
1
2 residues linked to Man
1
6(Man
1
3) Man
1
6(Man
1
3)Manß1
4GlcNAcß1
4GlcNAc-2AB (component e) instead of complex type sugar chains as found in other clones.
On the basis of these results, the structures of oligosaccharides on IgG1 produced by the wild type Pro-5 CHO cells and the glycosylation mutants Lec 2, Lec 8, and Lec 1 were elucidated and are summarized in Table I.
In vivo clearance of IgG1 with different carbohydrate structures
Mice injected intravenously with protein iodinated through the attached dilactotyramine moiety were sacrificed 3 or 6 days following injection, their organs were harvested, and the associated radioactivity was determined. The results are shown in Figure 3A and are expressed as the total radioactivity present in a particular organ compared to the total radioactivity present at the time of sacrifice. Values represent the average of two mice. For all of the proteins, there is significant residual radioactivity in the blood, skin, and muscle. For IgG1-Lec 2 and IgG1-Lec 1 there is also significant quantities of radioactivity present in the liver at days 3 and 6.
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This treatment of the data indicates that the majority of all of the antibodies irrespective of the structure of their attached carbohydrate is catabolized in the skin and muscle. However, the attached carbohydrate structure does influence the amount that is catabolized in the liver, and the liver serves as a major site for the catabolism of proteins bearing carbohydrate with the Lec 2 (with terminal galactose) or Lec 1 (with terminal mannose) structure.
We also investigated the distribution of the radioactivity in the body on a weight basis (Figure 4). When the weight of the organs is considered, the radioactivity is seen to be broadly distributed but with reduced accumulation on a weight basis in the stomach, intestine and muscle. Comparison of TCA precipitable (Figure 4B) and TCA soluble (Figure 4C) radioactivity suggests that catabolism occurs in all organs although some of the TCA soluble counts present in the blood, stomach intestine, and kidney may represent catabolic products that are in the process of being excreted.
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Discussion |
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CHO cells are frequently used for the production of recombinant human glycoproteins. They do not attach the immunogenic (1
3) galactose residue and the oligosaccharide structures obtained from CHO-produced proteins such as erythropoietin and tissue plasminogen activator closely correspond to those derived from the native protein (Smith et al., 1990
; Goochee et al., 1991
). The derivation of several CHO cell lines with well-characterized glycosylation mutants has yielded a useful system in which to produce and evaluate glycoproteins with defined carbohydrate constituents.
These studies have shown that the CHO expression system with its Lec mutants is suitable for producing IgGs with altered carbohydrate structure. IgG1-Lec 1 (high-mannose intermediate) has an oligomannose structure as expected. Antibodies produced in Lec 8 (agalactosyl) have the most homogeneous carbohydrate structure suggesting that Lec 8 would be an appropriate expression system for production of proteins when uniform carbohydrate structure is desired. Although multiple different glycoforms are present on IgG1-Pro-5 and IgG1-Lec 2, the latter two antibodies showed far less heterogeneous glycosylation than the 30 glycoforms reported for human serum immunoglobulin. Pro-5 was the only cell line capable of producing sialylated IgG. However, only 8% of the carbohydrate structure present on IgG produced by Pro-5 had terminal sialic acid, similar to the extent of sialyation seen on human IgG (Rademacher et al., 1985
). A higher percentage of IgG1-Lec 2 than of IgG1-Pro-5 bears carbohydrate with two terminal galactose residues while Pro-5produced IgG1 has more structures with two terminal GlcNAc residues than Lec 2produced IgG1. With the exception of the Lec 1produced antibodies, all carbohydrate structures present on the antibodies are seen on normal human immunoglobulins,
The rate at which a therapeutic agent is catabolized plays an important role in determining its efficacy. Although these studies have shown that the glycoform present on the Fc of the antibody molecule can influence it biolocalization the major sites of protein localization for all of the IgGs, 3 and 6 days after injection are the blood, liver, skin, and muscle. The blood represents a recirculating pool of intact protein. The catabolism of IgG occurs throughout the body. When the total amount of catabolism is considered, the skin and muscle catabolize the most, irregardless of the structure of the attached carbohydrate. These results are largely in agreement with those of Henderson et al. (1982), who found that rat IgG is catabolized at several sites in the body.
The liver also serves as a major catabolic site, however the structure of the attached carbohydrate plays a significant role in determining the amount of IgG that is catabolized there. Within the liver several receptors are candidates for IgG clearance. One, the asaialoglycoprotein receptor, binds terminal galactose residues of desialyated glycoprotein and mediates endocytosis and eventual degradation of these ligands. Seventy percent of the carbohydrate structures present on IgG1-Lec 2 contain terminal galactose residues, allowing it to be recognized and degraded through this receptor pathway. It is noteworthy that IgG1-Pro-5produced proteins exhibit decreased liver targeting; only 65% of the glycoforms have galactose residues with 8% having sialic acid. More IgG1-Pro-5 protein is seen in the liver on day 6 than on day 3; possibly removal of sialic acid occurs in the circulation. Another candidate receptor is the mannose binding receptor expressed on Kupffer and sinusoidal endothelial cells (Lennartz et al., 1987; Stahl, 1992
; Takahashi et al., 1998
). It would appear likely that IgG1-Lec 1 proteins are recognized by this receptor. Indeed, earlier studies (Wright and Morrison, 1994
) have shown that administration of yeast mannan delays the clearance of IgG1-Lec 1. The mannose receptor could also contribute to the clearance of IgG1-Lec 8 since a recent study has demonstrated the binding and uptake of agalactosyl IgG through the mannose receptor (Dong et al., 1999
). Virtually all radioactivity present in the liver at both day 3 and day 6 is TCA soluble.
Recently the neonatal Fc receptor (FcRn) responsible for transport of immunoglobulin across the neonatal rodent intestine has been proposed to play a role in the control of the catabolism of IgG. FcRn resembles a Class I MHC molecule in structure. In ß2-microglobulin (ß2-m) deficient mice which fail to express FcRn, the half-life of IgG is much shorter (Ghetie et al., 1996; Israel et al., 1996
). The expression of FcRn is widespread and includes the capillary endothelium (Story et al., 1994
; Blumberg et al., 1995
; Israel et al., 1996
, 1997; Leach et al., 1996
). IgGs lacking carbohydrate continue to be recognized by FcRn (Hobbs et al., 1992
). Thus, it may be expected that alterations in carbohydrate structure would not affect recognition by FcRn.
FcRn shows a pH-dependent binding exhibiting high affinity for IgG at pH 6 and low affinity at neutral pH (Raghavan et al., 1995). The concept has evolved that FcRn binds IgG that has been taken up in the fluid phase in the acidified endosome and diverts it from trafficking to the lysosome (Junghans and Anderson, 1996
). This model postulates a saturable receptor in the salvage pathway consistent with the observation that the half-life of IgG decreases with increasing serum concentration. In this model the cells involved in salvaging IgGs are also responsible for IgG breakdown. Our data support the presence of a recycling receptor since significant quantities of both TCA precipitable and TCA soluble radioactivity are present at sites likely to express this receptor.
When the amount of residual counts per gram is considered several organs would appear to play an active role in catabolism on a per weight basis. Especially prominent is the spleen. Within the spleen there would be macrophages bearing the mannose receptor, and as discussed previously these may play a role (Gross et al., 1988; Smedsrod et al., 1990
;). Accordingly, of the antibodies described in this study, IgG1-Lec 1 was targeted to and catabolized most efficiently in the liver and spleen. In addition, the spleen would have cells bearing Fc receptors. However, it is not clear that Fc receptors play an active role in determining catabolic rate since IgGs lacking carbohydrate, and therefore not recognized by Fc receptors, clear at rates similar to what is observed with IgGs that are recognized by Fc receptors (Tao and Morrison, 1989
). For the heart and lungs, it may be the vascular endothelium that is participating in catabolism. For the kidney, the radioactivity may represent degraded protein in the process of being eliminated.
These studies have confirmed that the Lec mutants of CHO cells attach glycoforms of the expected structures and provide a convenient expression system for the production of glycoproteins with defined glycoforms attached. We have found that antibodies are catabolized at many sites throughout the body. Although the amount catabolized at each site is influenced by the structure of the attached carbohydrate, the general pattern of catabolism remains similar for all of the glycoforms. Although we do not have direct information about which cells and receptors in these tissues are actually interacting with the antibodies, our results are consistent with FcRn and the mannose and asialoglycoproteins receptors contributing to the catabolism.
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Materials and methods |
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Materials
Diplococcal ß-galactosidase and ß-N-acetylhexosaminidase were purchased from Boehringer Mannheim (Mannheim, Germany). -Mannosidase specific for Man
1
2 linkage was purified from Aspergillus saitoi as described previously (Yamashita et al., 1980
).
Liberation of the N-glycans of IgG as oligosaccharides
Each IgG was subjected to 9 h hydrazinolysis as described previously (Takasaki et al., 1982). After N-acetylation, oligosaccharide fraction was labeled with 2-aminobenzamide (2-AB) using the 2-AB labeling kit (Oxford Glycosystems, Abingdon, UK) to obtain 2AB-labeled oligosaccharides.
Analytical methods
Anion-exchange column chromatography and paper chromatography were performed as described previously (Chiba et al., 1997). Amide column HPLC was carried on GlycoSep N (Oxford Glycosystems), and the acetonitrile/250 mM ammonium acetate (pH 4.0) ratio was changed linearly from 80:20 to 47:53 (v/v) over 132 min after injection at a flow rate of 1.0 ml/min at 30°C. Neutral oligosaccharides were subjected to HPLC using an LA-RCA 120 (Ricinus communis agglutinin 120) column (Seikagaku Co., Tokyo). After elution with 16 ml of 10 mM phosphate buffer, pH 7.4, 140 mM NaCl, (PBS), the bound oligosaccharides were eluted with a gradient of PBS containing 02 mM lactose, at a flow rate of 0.8 ml/min at 30°C.
Glycosidase digestion
Oligosaccharides were incubated with one of the following mixtures at 37°C for 18 h: diplococcal ß-galactosidase (10 mU) in 50 µl of 0.3 M citratephosphate buffer (pH 6.0); diplococcal ß-N-acetylhexosaminidase (8 mU) in 50 µl of 0.3 M citratephosphate buffer (pH 6.0); A.saitoi -mannosidase (0.15 µg) in 30 µl of 0.1 M acetate buffer (pH 5.0). One drop of toluene was added to all reaction mixtures to inhibit bacterial growth during incubation. Digestion was terminated by heating the reaction mixture in a boiling water bath for 3 min and product was analyzed by amide column HPLC.
Oligosaccharides
GlcNAcß12Man
1
6(GlcNAcß1
2Man
1
3)Manß1
4GlcNAcß1
4(Fuc
1
6)GlcNAc-2AB and Man
1
6(Man
1
3)Man
1
6(Man
1
3)Manß1
4 GlcNAcß1
4GlcNAc-2AB were prepared from human myeloma IgG (Endo et al., 1989
) and bovine pancreatic ribonuclease B (Liang et al., 1980
), respectively, by hydrazinolysis followed by labeling with 2AB as described above. Man
1
6(Man
1
3)Manß1
4GlcNAcß1
4 (Fuc
1
6)GlcNAc-2AB was obtained from GlcNAcß1
2Man
1
6(GlcNAcß1
2 Man
1
3)Manß1
4GlcNAcß1
4(Fuc
1
6)GlcNAc-2AB by diplococcal ß-N-acetylhexosaminidase digestion.
Synthesis of the residualizing label dilactitol tyramine (DLT)
Synthesis of the glycoconjugate dilactitol tyramine (DLT) was performed according to Strobel et al. (1985). Briefly, a 5-ml reaction mixture containing 130 mM tyramine, 1.3 M lactose, and 520 mM sodium cyanoborohydride (NaBH3CN; molar ratios of the reactants 1:10:4) was combined in 0.2 M potassium borate buffer, pH 9, and incubated overnight at 65°C. The glycoconjugate was diluted and then purified from the reactants by cation-exchange chromatography on Dowex 50-X2 (Bio-Rad). Fractions were eluted with 0.05 M ammonium acetate followed by 1 M ammonium acetate, pH 7. The amount of protein in the fractions was determined using A280 absorbance; the presence of carbohydrate was determined using the anthrone reaction. The molar ratio of sugar to tyramine was determined for each fraction, and those exhibiting a 2:1 ratio were pooled and concentrated by lyophilization. The DLT was dissolved in distilled water and the concentration estimated by absorbance at 280 nm, using an extinction coefficient of 1360 M1 as described previously (Strobel et al., 1985
). The concentration of DLT was then adjusted to a concentration of 100 mM and stored at 20°C.
Radioiodination and coupling of DLT to the antibodies
The proteins were labeled with Na[125]I (Amersham) using the Iodobead method (Pierce). Radiolabeled DLT (1 µM) was incubated at 37°C with 4 U galactose oxidase (Sigma), which converts DLT to the aldehyde form. Purified immunoglobulin (20 mg, resuspended in PBS buffer pH 7.4) was added to the aldehyde as well as 40 mM NaBH3CN and incubated for up to 2 h at 37°C. The iodinated antibody was then separated from the free conjugate by chromatography on Sephadex G-50 equilibrated with PBS 7.4 and 1% BSA (bovine serum albumin, Sigma). The final radiolabeled protein was
95% precipitable in 10% TCA.
Animals
Female BALB/c mice (3 months old, Taconic Farms, Germantown, NY) were fed water treated with 0.1 mg/ml potassium iodide for at least 1 week before proteins were injected. Groups of four mice were injected intravenously (tail vein) with equal amounts of iodinated antibody. To determine whole body radioactivity each mouse was placed, at regular intervals, in a NaI crystal with a model JS-5A scaler/ratemeter attached (Ronceverte, WV). Measurements were begun immediately after injection. After 3 days, half the mice from each group were sacrificed. The remaining mice were sacrificed 6 days post-injection.
Mice were weighed and counted immediately before sacrifice using ether anesthesia. The abdominal area was shaved so that skin samples could be removed. The animals were exsanguinated and the blood allowed to clot. Organs were rapidly dissected out, rinsed with ice-cold PBS, and placed in labeled, preweighed Eppendorf tubes in a dry ice-ethanol bath. Tissues were kept cold to minimize protein degradation. Radioactivity remaining in each organ was measured and analysis of precipitable protein was performed within 24 h.
Analysis of organ distribution of proteins
Processing of tissues was performed as described previously (Thorpe and Baynes, 1994). Tissues were homogenized and aliquots were incubated with an equal volume of 40% TCA. To estimate the percentage of the degraded protein in the sample radioactivity was measured in both the supernatant and pellet. Radioactivity in the supernatant was calculated as the percentage of the total radioactivity of both the supernatant and pellet.
For skin and muscle, the radioactivity in the recovered aliquots was measured. Total radioactivity was calculated by multiplying the radioactivity of the aliquots per gram tissue by total tissue weight, estimated at 18% or 45% of the total body weight, respectively (Moldoveanu et al., 1988). TCA precipitability of the protein was calculated as described above.
In all cases the calculated specific radioactivity associated with the organ or tissue was corrected by subtraction of the blood-borne radioactivity. Based on published records of blood volumes of organs in mice, expressed as ml/kg of body weight (Altman and Dittmer, 1971), these values were adjusted for the weights of the experimental animals and accordingly subtracted from the total radioactivity recovered from each organ.
Interpretation of results
An estimate of the overall distribution of the distribution of the protein in the body was calculated as a percentage of the remaining for each organ. The contribution of each organ or tissue to degradation (catabolism) of the protein was calculated by multiplying the percentage of the remaining dose recovered in each tissue by the acid-soluble fraction of radioactivity. This was expressed as the % dose catabolized/tissue. To estimate the relative catabolic efficiency of each tissue, each of these values was divided by the weight of the appropriate tissue
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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