The products from papain and pepsin hydrolyses of guinea-pig immunoglobulins γ1G and γ2G

1. The products from papain and pepsin hydrolyses of the guinea-pig immunoglobulins gamma(1)G and gamma(2)G were isolated and characterized with regard to molecular weight, amino acid composition, hexose content and antigenic specificity. 2. Fragments Fab and (Fab')(2) from immunoglobulins gamma(1)G and gamma(2)G have similar electrophoretic and antigenic properties, but show some class-specific differences in amino acid composition. 3. Three Fc fragments were obtained after papain digestion of immunoglobulin gamma(2)G, namely, fragment Fc dimer (mol.wt. 58000), fragment Fc monomer (mol.wt. 29000) and fragment Fc' (mol.wt. 8000). A single crystalline fragment, namely fragment Fc' (mol.wt. 11000), was isolated after papain digestion of immunoglobulin gamma(1)G. 4. Peptic digestion of immunoglobulins gamma(1)G and gamma(2)G releases C-terminal fragments, namely, fragments pFc', of similar molecular weight (13000) but different amino acid compositions and distinct antigenic specificities. 5. Digestion-time studies show that immunoglobulin gamma(1)G is far more susceptible to proteolysis than is immunoglobulin gamma(2)G and suggest that at least a proportion of molecules are split primarily at a site that liberates fragment gamma(1)Fc'.     

小鼠抗天花粉蛋白IgE型单抗的酶解及其Fab的制备

研究了Ｐａｐａｉｎ及Ｔｒｙｐｓｉｎ裂解小鼠抗天花粉蛋白ＩｇＥ单抗的条件及Ｆａｂ的制备。Ｐａｐａｉｎ和Ｔｒｙｐｓｉｎ两者都可产生Ｆ（ａｂ′）_２,分子量在１５０～１６０ｋＤ左右;经Ｐａｐａｉｎ裂解的主要产物中还有Ｆａｂ,分子量７２ｋＤ,可通过凝胶过滤获得纯的Ｆａｂ。而Ｔｒｙｐｓｉｎ裂解物经ＤＴＴ还原、碘乙酰胺烷化虽然也可得到Ｆａｂ′（ｔ）,但不易纯化;可见,要制备Ｆａｂ以采用Ｐａｐａｉｎ裂解为好,而制备Ｆ（ａｂ′）_２则可采用Ｔｒｙｐｓｉｎ裂解。这二个酶的裂解速度是Ｔｒｙｐｓｉｎ大于Ｐａｐａｉｎ。     

Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.     

Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain

IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.     

The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the `hinge' region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the `hinge' region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of `hinge' peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the `hinge' region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the `hinge' region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.     

Purification of recombinant chimeric B72.3 Fab' and F(ab')2 using streptococcal protein G.

Streptococcal protein G has been used extensively for the purification of antibodies using the interaction of the Fc region with protein G. Many antibodies also interact with protein G through a low-affinity binding site for the Fab region. The exploitation of this low-affinity interaction for the purification of Fab' fragments is described here. Chimeric mouse-human B72.3 Fab' and F(ab')2 fragments were expressed by CHO cells and purified from CHO cell supernatant using protein G-Sepharose. Since chimeric B72.3 Fab' bound weakly to the protein G-Sepharose it could be separated from F(ab')2 and eluted with a pH 7 wash whereas B72.3 F(ab')2 required elution at pH 2. Both Fab' and F(ab')2 were recovered with full immunoreactivity and could be further purified using gel-filtration chromatography to greater than 99% purity. This method allows the simple purification of directly expressed Fab' or F(ab')2 fragments from CHO cell supernatant.     

Proton nuclear magnetic resonance study on the dynamics of the conformation of the hinge segment of human G1 immunoglobulin

A proton nuclear magnetic resonance (NMR) study is reported for the dynamics of the conformation of the hinge segment of human G1 immunoglobulin. The hinge fragment (Thr223-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu234)2 was obtained by tryptic digestion of F(ab')2, a peptic fragment of IgG1. Comparisons of the NMR results obtained for the hinge fragment with those for the intact IgG1 and its fragments led us to conclude that a significant change in conformation of the segment preceding the disulfide-linked Cys-Pro-Pro-Cys core is induced when the Fab portion is cleaved off and the presence or absence of the Fc portion affects very little, if any, of the conformation of this part of the hinge. On the basis of the present NMR results along with those which we have obtained previously using the intact IgG1 and its fragments, it was concluded that the conformation of the segment preceding the Cys-Pro-Pro-Cys core of the intact IgG1 can be maintained only when it is flanked by the Fab portion and the Cys-Pro-Pro-Cys core. An X-ray crystallographic study [Marquart, M., Deisenhofer, J., Huber, R., & Palm, W. (1980) J. Mol. Biol. 141, 369-392] showed that segment Cys-220-Thr-225 forms a one-turn helix with little inherent stability. Upon loss of Fab or Fc, residual segments of the hinge would become too short to form the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thioether-bonded constructs of Fab'gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab'gamma from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab')(n) core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab(2)Fc(2) (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab(3)Fc(2), which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab' in two reduced forms, Fab'(-sulfhydryl (SH))(5) and Fab'-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab')(2) fragment of IgG. Fab'-SH is obtained by disulfide-interchange reactions on Fab'(-SH)(5), whereby the gamma-light SS is reconstituted, an unusual intrachain SS forms in the gamma-chain hinge, and one hinge SH remains. F(ab')(2) and F(ab')(3) cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fcgamma modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab(2)Fc(2) yielded a t(1/2) similar to that of human IgG1, although the serum Fab(2)Fc(2) revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.     

Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

Production of humanized Fab fragment against human high affinity IgE receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (Fc epsilonRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human Fc epsilonRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human Fc epsilonRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and Fc epsilonRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M

Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgM(p) (Inman & Hazen, 1968). In the presence of mercaptans, IgM(p) partially dissociated into Fc(mu)-like and Fab(mu) fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with (14)C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgM(p) fractions. IgM and IgM 7S subunit (IgM(s)) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgM(p) by activating adsorbed papain.     

Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Characterization of two species of Fc fragments obtained from rat IgG2a by prolonged papain digestion.

Prolonged papain digestion of rat IgG2a produced two molecular species of Fc fragments, termed Fc(I) and Fc(II). Studies by gel filtration chromatography and polyacrylamide gel electrophoresis in SDS/urea indicated that the two subunit polypeptide chains in each Fc preparation were associated by non-covalent bonds only. By analytical ultracentrifugation Fc(I) was found to have a m.w. of 47,100 and a sedimentation coefficient of 4.08S. Fc(II) had a m.w. of 39,800 and a sedimentation coefficient of 3.83S. The m.w. for the subunit chains of Fc(I) and Fc(II) were 25,300 and 20,300, respectively, as determined by analytical ultracentrifugation under dissociating conditions. Calculation of the frictional coefficient ratios indicated that both Fc fragments possessed compact globular structures. The difference in size between these two Fc fragments probably was due to a loss of some carboxy-terminal residues in Fc(II). Both Fc fragments possessed nearly identical amino-terminal amino acid sequences. Papain cleavage occurred primarily between residues 233/234 and 234/235. The carbohydrate compositions of the two species of Fc fragments were similar. It was concluded that under acid and reducing conditions papain cleavage of rat IgG2a occurred to the carboxy-terminal side of the hinge region. Prolonged papain digestion led to secondary attack in the carboxy-terminal end of the CGAMMA3 domain at an unidentified site, or sites, producing a stable second species of Fc fragments.     

Region-selective labeling of antibodies as determined by electrospray ionization-mass spectrometry (ESI-MS)

The electrospray ionization-mass spectrometry (ESI-MS) analysis of three sets of monoclonal antibody-acridinium-9-carboxamide conjugates is described. The conjugates (nine total) were enzymatically digested using papain and the resulting fragments [Fc heavy chain, Fab, or F(ab')(2)] were analyzed using liquid chromatography/ESI-MS. The average number of labels per fragment were calculated using Sigma nx%, where n is the number of acridinium molecules covalently bound to the fragment and x% is the percent relative area of the corresponding peaks in the mass spectrum. When these values were normalized against the molecular weight of their respective region, antibody-dependent labeling patterns were observed. For antibodies T (anti-L-T(4)) and F (anti-FITC), there was a preference for conjugation of the Fab region over the Fc region. For antibody B (anti-biotin), the trend was reversed.     

Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized

Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment^1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)₂ fragment, which contains both antigen-binding sites. Based on this information, some investigators^6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG₂ subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG₂ subclass so far determined has approximately 95% homology with that of human IgG₁ and IgG₄ subclasses reported by others^9–15.     

Comparative analysis of the adjuvant action of fab-fragments of normal rabbit IgG obtained using pepsin and papain]

The capacity of Fab fragments of normal rabbit IgG to enhance the immune response to sheep erythrocytes in the homologous recipients was determined by the structure of the C-terminal part of the heavy chain Fd fragment. This followed from the fact that pepsin F(a')2 and Fab' fragments enhanced considerably the hemagglutinin production and proliferation of the antibody-forming cells in the spleen, whereas papain Fab fragments, used in the same dose as the pepsin fragments, possessed only negligible adjuvant activity. As shown the adjuvant activity of pepsin and papain fragments displayed an inverse correlation with the titres and papain homoreactants in the sera of the homologous recipients. The data obtained suggested that the target cells for Fab fragments were lymphocytes carrying cytophilic homoreactants as receptors for the fragments.     

Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies.

Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.     

Asymmetrically glycosylated IgG isolated from non-immune human sera

When human IgG or its F(ab')2 fragment purified from a pool of non-immune sera was passed through a Con A-Sepharose column, 12% of the molecules bound to concanavalin A. While 44% of Fab' and 72% of Fd' fragments obtained from F(ab')2 retained by concanavalin A and eluted with methyl alpha-D-mannoside bound to concanavalin A, the Fab' and Fd' fragments obtained from non-retained F(ab')2 and the L chains and Fc fragments did not interact with the lectin. Only Fd' fragment obtained from the F(ab')2 retained by concanavalin A inhibited the fixation of guinea-pig erythrocytes to concanavalin A. These results are similar to those previously observed for IgG antibodies of different animal species and indicate that partial asymmetric glycosylation is a general phenomenon that is not restricted exclusively to IgG molecules with known specificity.