- Email: [email protected]
Engineering monoclonal antibodies to determine the structural requirements for complement activation and complement mediated lysis
MolecularImmunology, Vol. 28,No. 12,Pp. 1361-1368, 1991 Printedin Great Britain.
0161-5890/91 $3.00+ 0.00 PergamonPressplc
ENGINEERING MONOCLONAL ANTIBODIES TO DETERMINE THE STRUCTURAL REQUIREMENTS FOR COMPLEMENT ACTIVATION AND COMPLEMENT MEDIATED LYSIS and TERJE E. MICHAELSEN~
INGER SANDLIE*?
*Department of Biology, University of Oslo, Norway and SDepartment National Institute of Public Health, Oslo, Norway (Received
1 December 1990)
INTRODUCTION The classical complement
pathway
tem generating
of potent
a variety
is a cascade sysbiological
mol-
ecules. The pathway is triggered by the interaction the first complement gen complexed Cls,
IgG. Cl is composed
and the Clq
domain
of
protein complex,
of
Cl, with anti-
of Clq,
Clr and
subunit interacts with the second
the heavy
chain
(CH2)
on IgG.
of Immunology,
The
residues
Glu 318, Lys 320 and Lys 322 on CH2 are involved in the binding (Duncan and Winter, 1988). This core binding motif is conserved in the four human IgG subclasses, both the lytic and the nonlytic molecules. Therefore further structural determinants must be involved in the lysis mechanism. Clq has a molecular weight of approximately 460,000, and has the appearance of a “bunch of tulips” (Reid and Porter, 1981). The molecule is multivalent in its binding to IgG, and binding to monomeric IgG is weak (Ka 104M-‘). When several IgGs bind to multiple epitopes on an antigenic surface, the resulting aggregation of IgG molecules allows the binding of two or more tulip heads leading to a tight binding (Ku lOaM-‘) which is necessary for the activation process to proceed. It is possible that an exact alignment of antibodies and Clq is required for full activation, and also that some degree of flexibility in the molecules is necessary. Clearly, this would reduce the stringency of the steric requirements when Clq binds to an array of IgG molecules. Some flexibility is detected in the Clq molecule (Poon et al., 1983), and several modes of flexibility have been demonstrated for IgG molecules (Nezlin, 1990). X-ray diffraction analysis of whole IgG crystals have been characterized by a lack of electron density associated with part of the hinge and the whole of Fc, and the phenomenon has been attributed to hinge flexibility (Huber et al., 1976). The hinge can be tAuthor to whom correspondence should be addressed at: Department of Biology, University of Oslo, P. 0. Box 1066, N-0316 Oslo 3, Norway.
divided into three regions, the upper, middle and lower hinge (Table 1). The upper hinge has been defined by Burton (1987) as the number of amino acids between the end of the first domain of the heavy chain (CHl) and the first cysteine forming an interheavy cysteine disulfide bridge and a high content of proline. The lower hinge connects the middle hinge to the CH2 domain. The information to date concerning the conformation of the hinge indicates that it has the characteristics of a polyproline helix (Johnson et al., 1975; Endo and Arata, 1985; Ito and Arata, 1985). This is a relatively rigid, rod-like double-stranded structure. All the IgG middle hinge sequences have similar polyproline cores (Table 1) and probably adopt the same polyproline structure. The length of the middle hinge however, varies widely among the human IgG subclasses, and is 50 amino acids for IgG3, and 5 amino acids for IgGl. The middle hinge can have several functions; it contains the cysteines participating in disulfide bridges and thus contains the residues keeping the two CH2 domains together in the N-terminal end (Michaelsen, 1976), the rod-shaped core provides spacing or distance between the antigen binding and effector domains, and also the polyproline double helix possibly maintains an appropriate spatial relationship between these two parts of the molecule. The lower hinge may be the site for Fc motion relative to Fab. There is little amino acid variation in the lower hinges of the human IgGs. However, one murine subclass, namely IgGl, has a rather short lower hinge lacking the three amino acids usually found in position 233, 234 and 236.
UPPER HINGE LENGTH, SEGMENTAL FLEXIBILITY AND COMPLEMENT
ACTIVATION
Dangl et al. (1988) have generated a family of nine IgG molecules with identical antigen-binding sites recognizing the DNS hapten and heavy chain constant regions of human, mouse and rabbit origin, 1361
1362
INGER SANDLIE and TERJE E. MICHAELSEN
Table 1. Comparison of binge sequences of human immunoglobulins
Human IgGl Human IgG2 Human IgG3 Human IgG3M 15 Human IgG4 Mouse IgGl Mouse IgG2a
Upper hinge 216
Middle
EPKSCDKTHT ERK ELKTPLGDTTHT EPKS ESKYGPP VPRDCG EPRGPTIKP
CPPCP CCVECPPCP CPRCP (EPKSCDTPPPCPRCP), CDTPPPCPRCP CPSCP CKPCICT CPPCKCP
respectively. The segmental flexibility of the molecules was measured and found to correlate with the length of the upper hinge. Human IgGl and IgG3 which have upper hinges of 10 and 12 amino acids, respectively, showed high flexibility, whereas IgG2 and IgG4 have short upper hinges (3 and 7 amino acids) and were relatively rigid. A long upper hinge will allow the angle between the Fab arms to vary widely, whereas a short upper hinge allows for little Fab arm motion. This hinge flexibility allows divalent recognition of variably spaced antigenic determinants. The ability of Ig molecules to change their conformation from Y to T shape (Nezlin, 1990), greatly facilitates the capacity of antibodies to crosslink antigens. Segmental flexibility has been correlated with complement activation of IgG. Of the nine different IgG molecules studied by Dangl et al. (1988) the flexible antibodies were able to fix complement most effectively. To study the proposed association between hinge length, flexibility and effector function, we made mutants of mouse/human chime& IgG3 antibodies varying the length of the upper hinge and the core hinge, while the rest of the molecule was kept unaltered (Sandlie et al., 1989). Thus, the effect of manipulating the hinge could be studied independently of any effects due to isotype differences in CHl, CH2 and CH3. The antibodies have specificity for the NIP hapten. Five different deletion mutants were constructed as shown in Fig. 1, deleting one or more hinge exons. The resulting antibodies have hinges of 47, 45, 32, 15 and 0 amino acids respectively. The length of the upper hinges of mutant M47 and M32 are identical to that of IgG3 wild type, namely 12 amino acids, whereas the upper hinge of M45 and M15, where the first exon is deleted, consists of 4 amino acids. M47, M45, M32 and Ml5 all reacted with IgG3 hinge-specific antibodies, thus the hinge epitopes were mainly retained. The new CHI H
hl
h2 h3 I4
e$ b ML7 ML5 _M32 Ml5 MO
6
CH2
CH3 1
b
5
l&Q
Fig. 1. Restriction map of the y3 gene. Exons are shown as boxes. H, Hind III; Bg, Bgl II; P, Pst I; S, Sph I. Lines indicate
deletions
in the gene.
hinge
Lower hinge 238 APELLGGP APPVAGP APELLGGP APELLGGP APEFLGGP VPSEVS APNLLGGP
antibodies were tested for their ability to activate complement. M45 and Ml5 activated human complement equally well or better than wildtype IgG3 (Sandlie et al., 1989; Michaelsen et al., 1990~). Both M45 and M15, having upper hinge of four amino acids, activated complement. M45 was equally efficient as wild type IgG3, while Ml5 was more efficient. Therefore, neither a long upper hinge nor a long total hinge is necessary for complement activation. It seems that the long hinge of IgG3 rather than enhancing complement activation by this subclass, downregulates its activation potential. Tan et al. (1990) made similar IgG3 mutants with specificity for the DNS hapten. Segmental flexibility of the antibodies were measured, and found to be low for the mutant with the first hinge exon deleted. These rigid antibodies were also shown to activate complement efficiently. Thus, complement activation does not depend on a high degree of segmental flexibility. However, in several species there is a correlation between segmental flexibility of naturally occurring antibodies and ability to fix complement. Possibly, the organisms have an advantage when the molecules most active in complement activation are able to do extensive Fab-arm waving, and thereby bind variably spaced epitopes, while the nonlytic molecules are limited in this respect.
THE AMINO ACID SEQUENCE OF THE HINGE MODULATES THE ABILITY OF I& TO ACTIVATE COMPLEMENT
IgG4 is completely inactive in complement activation. It is a rigid molecule with a short upper hinge, its core hinge contains the central common hinge sequence (cysteine-proline-proline-cysteine), and the lower hinge is identical to that of IgGl and IgG3 except for a phenylalanine substituted for leucine in position 234. To determine whether the amino acid sequence of the genetic IgG4 hinge is responsible for its lack of effector function, we mutated the hinge exon of Ml5 by in vitro mutagenesis to become identical to that of IgG4. Instead of losing effector function, this IgG3-derived molecule (M15C, Fig. 2) activated complement even better than IgG3 Ml5 (Fig. 3) (Sandlie et al., submitted). Therefore, the amino acid sequence of the IgG4 hinge alone can not be the reason why IgG4 does not activate complement. Chimeric anti-DNS IgG3 molecules with the hinge of IgG4 were also made by Tan et al. (1990) as well
1363
Complement activation and complement mediated lysis Iw3wt IHJ-I-HI
MIS
s
M15C
F
MlSD
s
M15E
B
M15F
B
M15G
0
IgG3
-
IgGG
Fig. 2. Schematic drawing of the IgG3 derived antibody genes. Exons are shown as boxes. as IgG4 molecules with the hinge of IgG3. Functional studies performed on these antibodies also demonstrate that the primary structure of the IgG4 hinge is not the reason why IgG4 does not activate complement. However, in this work the IgG3 mutant with an IgG4 hinge was secreted as a mixture of H2L, molecules and HL half-molecules due to lack of appropriate disulfide bridging, and complement activation could not be measured accurately. Schneider et al. (1988) measured the segmental flexibility of genetically engineered mouse IgGl/IgG2 hybrids with specificity for DNS. It was demonstrated that proper “matching” of the iv-terminal part of the CHl domain and the hinge was important for the flexibility of the IgG molecule. The
N-terminal part contains a loop formed by residues 131-139 in CHl (EU numbering). It has previously been demonstrated by X-ray crystallography of human IgGl Kol that this loop is very near to the hinge (Marquart et al., 1980). The only differences between IgG4 and IgG3 in the N-terminal part of CHl are in residues 137 and 138 (Fig. 4). We mutated these two residues in CHl of IgG3 to become identical to those of IgG4 (M15D, Fig. 2). Complement activation was measured and found to be equal to that of wild-type IgG3 (Fig. 3) suggesting that of the mutations tested, those affecting the hinge are the most important in modulating this effector function. Segmental flexibility of the molecules have not been studied. When this mutation was introduced into the IgG3 molecule with the hinge of IgG4 (Ml5E, Fig. 2), complement activation improved relative to M 15D (Fig. 3). We also made molecules which are IgG4-like in CHl and the hinge, and IgG3-like in CH2 and CH3 (Ml5G, Fig. 2). These molecules activate complement as efficiently as Ml5 These results are unexpected, since Fc fragments from human IgG4 have been shown to bind Clq with an affinity comparable to that of the corresponding fragment from IgGl (Isenmann et al., 1975). The results from Isenmann et al. (1975) were interpreted to suggest that IgG4 Fab may prevent Clq binding by blocking the access to the IgG4 Fc Clq binding site. We have shown that Fab-hinge from IgG4 positively modulates Clq binding on IgG3. It remains to be shown whether structural features in Fc of IgG4 alone is responsible for the lack of Clq binding observed for IgG4, or whether an interplay between Fab and Fc is involved.
Ch
Absorbance
1.8 , t 1.6
1
1.4
t
+k
Ml5
--A--
lgG3
-a-
M15C
-A-
M15D
-t,-
M15E
+
M15F
-
M15G
wt
t 1.2
1
t
’ t 0.8 t 0.6 0.4 0.2 0 3.7
11.1
33.3
100
300
900
Concentration(ng/ml)
Fig. 3. EIA analysis of complement activation. The experiments were performed in microtiter wells coated with NIP-BSA conjugate. Serial dilutions of chimeric antibodies were added to the wells. Normal fresh serum diluted 1: 30 in isotonic Verona1 buffer was used as complement source, and complement activation measured by the binding of rabbit anti-Clq antibodies to the wells. MlMM
28,12-0
INGER SANDLIE and
1364
TERJEE. MICHAELSEN
CML
Concentratii
MPG.-FOb'
I
of chime& antibodes ng/ml
Fig. 6. Complement mediated lysis of hinge mutants of IgG3. The molecules tested were wild type IgG3 (17-IS-1%15), a variant with 47 amino acid hinge (17-1515), a variant with 32 amino acid hinge (17-15), a variant with a 15 amino acid hinge (1 S), a variant with 45 amino acid hinge (15-15-15) and a variant without hinge (0). IgGl is also included for comparison.
THE CHZ DOMAIN CARRIES FEATURES CRITICAL FOR COMPLEMENT ACTIVATION IN ADDITION TO THE Clq BINDING MOTIF
Clackson and Winter (1989) have created hybrids of lytic and non-lytic murine antibodies with specificity for the NIP hapten, and have shown that the CH2 exon of the IgG2b antibody active in complement-mediated lysis can confer lytic ability when replacing the CH2 exon on the usually inactive IgGl. This also argues against the hypothesis that the length of the upper hinge determines the ability of an antibody to activate complement. The lower hinge of mouse IgGl is rather short, and lacks the three amino acids usually found in positions 233, 234 and 236. Human IgG4 also has an abnormality in the lower hinge, namely the phenylalanine in position 234. The IgG subclasses have evolved independently in the two species, mouse and man, and IgG4 is not the human equivalent to mouse IgGl (Callard and Turner, 1990). Mouse IgGl can be shown to display a slight Clq binding ability at some experimental conditions, while IgG4 totally lacks complement activation ability regardless of conditions (Michaelsen et al., 19906). Therefore the structures rendering these two antibodies non-lytic may be different. Comparing the amino acid sequence of IgG3 and IgG4 in Fc, six amino acids differ in CH2 and three in CH3, not counting the residues that are unique to IgG3, and where IgGl, IgG2 and IgG4 use the same residue. In CH2, these are all located to the upper half of the globular domain structure, and several are located in hinge proximal bends formed between p-strands (Fig. 5) (Deisenhofer, 1981). This area of the globular CH2 domain may prove to be of major importance for Clq binding and complement activation. The carbohydrate bound to Asn 297 in CH2 is necessary for Clq-binding (Duncan and Winter, 1988) as aglycosylated antibodies produced by an Asn/Ala amino acid substitution at residue 297 show a three-fold lower association constant for Clq and
does not activate C 1. Studies by Lund et al. (in press) suggest that aglycosylation results in very localized structural changes in the lower hinge. It is suggested that Clq interacts both with the “core” binding motif and the lower hinge, and that aglycosylation results in loss of accessibility to the hinge. Tight Clq binding is however not sufficient for complement activation and complement lysis to occur, since both wild type antibodies and mutant variants possessing Clq binding activity can be non-lytic (Duncan and Winter, 1988; Bindon et al., 1988).
COMPLEMENT
MEDIATED
LYSIS
When comparing the four human IgGs in complement mediated lysis (CML), we found that IgG3 was the most efficient in complement activation, as well as lysis (Michaelsen et al., 1990b; Garred et al., 1989). To measure CML, SRBC was incubated with Na “0, and sensitized with the hapten NIP antigen as follows: NIP was conjugated to rabbit anti-SRBC Fab as described (Michaelsen et al., 1990a). Both the amount of Fab fragment that was bound to a total of 10’ SRBC and the number of hapten molecules that were conjugated to each Fab fragment was varied. After incubation at 37°C the cells were washed and diluted to 2-3 x 10’ cells/ml, Serial dilutions of chimeric anti-NIP antibodies were added to the target cell solution before human serum was added. After a 30-min incubation at 37°C the amount of released “Cr was measured with a gamma counter. The activity of the different subclasses could thus be measured varying the epitope concentration and also the epitope density, or “patchiness” on the target cells. It was indeed found that the activity of the different subclasses do depend on these features. IgGl, however active at high epitope concentration and epitope patchiness, was found to have a low activity at low epitope concentration and low epitope patchiness (Fig. 6). IgG3 was the most active at a wide range of conditions tested. It is noteworthy that the short hinge mutant Ml5 proved more lytic than
Complement activation and complement mediated lysis
Fig. 4. Ribbon through the CHl domain from human IgGl (Marquart et al., 1980). Residues which differ between IgG3 and IgG4 in a hinge proximal loop are indicated with red balls. The heavy chain is yellow, the light chain is green. The start of the hinge is localized by an arrow.
1365
1366
INGER
SANDLIEand TERJEE. MICHAEUEN
Fig. 5. a-Carbon trace of human Fc (Deisenhofer, 1981). Residues which differ between IgG3 and IgG4 are marked with red balls. The Clq binding site in CH2 is marked with yellow balls.
Complement activation and complement mediated lysis Table 2. Antibody induced complement mediated lysis Antibody tested
Antibody concentration needed for 50% lysis (n&l)
IgG3 wild type Ml5 M15C
36 4 0.6
The target cells (SRBC) were sensitized with NIP coupled to Fab anti-SRBC. The hapten: Fab ratio was 15:1, and 400 ng hapten was added to lo8 target cells.
IgG3 wildtype (Michaelsen et al., 1990a), and that the mutant M15C was even more lytic (Table 2). Work by others employing the same chimeric mouse/human antibodies with antigen specificity for NIP (Bruggemann et al., 1987; Bindon et al., 1988) suggest that IgGl is superior in CML due to higher activity at a later stage in the complement cascade, namely C4 activation. Our work does not confirm these findings, and the discrepancy probably reflects the differences in the in vitro experimental conditions employed. Experiments in vivo will eventually determine how efficient the various antibodies are in lysing target cells
CONCLUSIONS
Recent work with genetically engineered monoclonal antibodies show that the hinge region modulates complement activation. However, neither a long upper hinge nor high segmental flexibility is necessary for efficient complement activation. Also, structures in the lower hinge or hinge proximal residues in the globular CH2 domain seem to be important. Acknowledgements-The help of Dr Christian Ramming to create the computer graphics is greatly appreciated. The work was supported by grants from The Norwegian Cancer Society.
REFERENCES
C. I., Hale G., Bruggemann M. and Waldmann H. (1988) Human monoclonal IgG isotypes differ in comp-
Bindon
lement activating function at the level of C4 as well as Clq. J. exp. Med. 168, 1277142. Bindon C. I., Hale G. and Waldman H. (1990) Complement activation by immunoglobulin does not depend solely on Clq binding. Eur. J. Immun. 20, 277-281. Bruggemann M., Williams G. T., Bindon C. I., Clark M. R., Walker M. R., Jefferis R., Waldman H. and Neuberger M. S. (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J. exp. Med. 166, 1351-1361. Burton D. R. (1987) Structure and function of antibodies. In Molecular Genetics of Immunoglobulin (Edited by Calabi F. and Neuberger M. S.), p. 1. Elsevier, Amsterdam. Callard R. E. and Turner M. W. (1990) Cytokines and Ig switching: evolutionary divergence between mice and humans. Zmmun. Today 11, 200-203. Clackson T. and Winter G. (1989) ‘Sticky feet”-directed mutagenesis and its application to swapping antibody domains. Nucl. Acids Res. 17, 10163-10170. Dangl J. L., Wensel T. G., Morrison S. L., Stryer L., Herzenberg L. A. and Oi V. T. (1988) Segmental flexibility and complement fixation of genetically engineered
1367
chimeric human, rabbit and mouse antibodies. EMBO J. 7, 1989-1994. Deisenhofer J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9 and 2.8 8, resolution. Biochemistry 20, 2361-2370. Duncan A. R. and Winter G. (1988) The binding site for Clq on IgG. Nature 332, 738-740. Endo S. and Arata Y. (1985) Proton nuclear magnetic resonance study of human IgGl and their proteolytic fragments: structure of the hinge region and effects of a hinge region deletion on internal flexibility. Biochemistry 24, 156lLl568. Garred P., Michaelsen T. E. and Aase A. (1989) The IgG subclass pattern of complement activation depends on epitope density, antibodyand complement concentration. Stand J. Immun. 30, 379-382. Huber R., Deisenhofer J., Colman P. M., Masaak M. and Palm W. (1976) Crystallographic structure studies of an IgG molecule and a Fc fragment. Nature 264, 415-420. Isenmann D. E.. Dorrineton K. J. and Painter R. H. (1975) The structure’and function of immunoglobulin do&&n: II. The importance of interchain disulfide bonds and the possible role of molecular flexibility in the interaction between immunoglobulin and complement. J. Immun. 114, 172661729. Ito W. and Arata Y. (1985) Proton nuclear magnetic study of the dynamics of the conformation of the hinge segment of human Gl immunoglobulin. Biochemistry 24, 6467-6474. Johnson P. M., Michaelsen T. E. and Scopes P. M. (1975) Conformation of the hinge and various fragments of human IgG3. &and. J. Immun. 4, 113-119. Lund J., Tanaka T., Takahashi N., Sarmay G., Araka Y. and Jefferis R. (1990) A protein structural change in aglycosylated IgG3 correlates with loss of human FcRI and human FcRIII binding and/or activation. Molec. Immun. (in press). Marquart M., Deisenhofer J., Huber R. and Palm W. (1980) Crystallographic refinement and atomic models of the _.._ - _ intact immunoglobulin molecule Kol and its antigenbinding fragment at 3.0 8, and 1.9 8, resolution. J. molec. Biol. 141, 3699391. Michaelsen T. E. (1976) Indications that the CH2 homology region is not a regular domain. Stand. J. Immun. 5, 1123-I 128. Michaelsen T. E., Aase A., Westby C. and Sandlie I. (1990a) Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons. &and. J. Immun. 32, 517-528. Michaelsen T. E., Garred P. and Aase A. (199Ob) Human IgG subclasses induce complement-mediated cytolysis differently upon changes in antibody affinity, complement concentration and epitope density. Eur. J. Immun. (in press). Nezlin R. (1990) Internal movements in immunoglobulin molecules. Adu. Immun. 48, l-39. Poon P. M., Schumaker V. N., Phillips M. L. and Strang C. J. (1983) Conformational and restricted segmental flexibility of Cl, the first component of human complement. J. molec. Biol. 168, 563-577. Reid K. E. M. and Porter R. R. (1981) The proteolytic activation systems of complement. A. Rev. Biochem. 50, 433-464. Sandlie I., Aase A., Westby C. and Michaelsen T. E. (1989) Clq binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids. Eur. J. Immun. 19, 1599-1603. Sandlie I., Norderhaug L., Brekke 0. H. A., Bremnes B., Sandin R., Aase A. and Michaelsen T. E. The amino acid sequence of the hinge modulates the lytic ability of IgG. Submitted.
1368
INGERSANDLIE and TERJEE. MICHAELSEN
Schneider W. P., Wensel T. G., Stryer L. and Oi V. T. (1988) Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility. Proc. natn.
Acad. Sci. U.S.A. 85, 2509-2513.
Tan L. K., Shopes R. J., Oi V. T. and Morrison S. L. (1990) Influence of the hinge region on complement activation, Clq binding, and segmental flexibility in chimeric human immunoglobulins. Proc. natn. Acad. Sci. U.S.A. 87,
162-166.
0161-5890/91 $3.00+ 0.00 PergamonPressplc
ENGINEERING MONOCLONAL ANTIBODIES TO DETERMINE THE STRUCTURAL REQUIREMENTS FOR COMPLEMENT ACTIVATION AND COMPLEMENT MEDIATED LYSIS and TERJE E. MICHAELSEN~
INGER SANDLIE*?
*Department of Biology, University of Oslo, Norway and SDepartment National Institute of Public Health, Oslo, Norway (Received
1 December 1990)
INTRODUCTION The classical complement
pathway
tem generating
of potent
a variety
is a cascade sysbiological
mol-
ecules. The pathway is triggered by the interaction the first complement gen complexed Cls,
IgG. Cl is composed
and the Clq
domain
of
protein complex,
of
Cl, with anti-
of Clq,
Clr and
subunit interacts with the second
the heavy
chain
(CH2)
on IgG.
of Immunology,
The
residues
Glu 318, Lys 320 and Lys 322 on CH2 are involved in the binding (Duncan and Winter, 1988). This core binding motif is conserved in the four human IgG subclasses, both the lytic and the nonlytic molecules. Therefore further structural determinants must be involved in the lysis mechanism. Clq has a molecular weight of approximately 460,000, and has the appearance of a “bunch of tulips” (Reid and Porter, 1981). The molecule is multivalent in its binding to IgG, and binding to monomeric IgG is weak (Ka 104M-‘). When several IgGs bind to multiple epitopes on an antigenic surface, the resulting aggregation of IgG molecules allows the binding of two or more tulip heads leading to a tight binding (Ku lOaM-‘) which is necessary for the activation process to proceed. It is possible that an exact alignment of antibodies and Clq is required for full activation, and also that some degree of flexibility in the molecules is necessary. Clearly, this would reduce the stringency of the steric requirements when Clq binds to an array of IgG molecules. Some flexibility is detected in the Clq molecule (Poon et al., 1983), and several modes of flexibility have been demonstrated for IgG molecules (Nezlin, 1990). X-ray diffraction analysis of whole IgG crystals have been characterized by a lack of electron density associated with part of the hinge and the whole of Fc, and the phenomenon has been attributed to hinge flexibility (Huber et al., 1976). The hinge can be tAuthor to whom correspondence should be addressed at: Department of Biology, University of Oslo, P. 0. Box 1066, N-0316 Oslo 3, Norway.
divided into three regions, the upper, middle and lower hinge (Table 1). The upper hinge has been defined by Burton (1987) as the number of amino acids between the end of the first domain of the heavy chain (CHl) and the first cysteine forming an interheavy cysteine disulfide bridge and a high content of proline. The lower hinge connects the middle hinge to the CH2 domain. The information to date concerning the conformation of the hinge indicates that it has the characteristics of a polyproline helix (Johnson et al., 1975; Endo and Arata, 1985; Ito and Arata, 1985). This is a relatively rigid, rod-like double-stranded structure. All the IgG middle hinge sequences have similar polyproline cores (Table 1) and probably adopt the same polyproline structure. The length of the middle hinge however, varies widely among the human IgG subclasses, and is 50 amino acids for IgG3, and 5 amino acids for IgGl. The middle hinge can have several functions; it contains the cysteines participating in disulfide bridges and thus contains the residues keeping the two CH2 domains together in the N-terminal end (Michaelsen, 1976), the rod-shaped core provides spacing or distance between the antigen binding and effector domains, and also the polyproline double helix possibly maintains an appropriate spatial relationship between these two parts of the molecule. The lower hinge may be the site for Fc motion relative to Fab. There is little amino acid variation in the lower hinges of the human IgGs. However, one murine subclass, namely IgGl, has a rather short lower hinge lacking the three amino acids usually found in position 233, 234 and 236.
UPPER HINGE LENGTH, SEGMENTAL FLEXIBILITY AND COMPLEMENT
ACTIVATION
Dangl et al. (1988) have generated a family of nine IgG molecules with identical antigen-binding sites recognizing the DNS hapten and heavy chain constant regions of human, mouse and rabbit origin, 1361
1362
INGER SANDLIE and TERJE E. MICHAELSEN
Table 1. Comparison of binge sequences of human immunoglobulins
Human IgGl Human IgG2 Human IgG3 Human IgG3M 15 Human IgG4 Mouse IgGl Mouse IgG2a
Upper hinge 216
Middle
EPKSCDKTHT ERK ELKTPLGDTTHT EPKS ESKYGPP VPRDCG EPRGPTIKP
CPPCP CCVECPPCP CPRCP (EPKSCDTPPPCPRCP), CDTPPPCPRCP CPSCP CKPCICT CPPCKCP
respectively. The segmental flexibility of the molecules was measured and found to correlate with the length of the upper hinge. Human IgGl and IgG3 which have upper hinges of 10 and 12 amino acids, respectively, showed high flexibility, whereas IgG2 and IgG4 have short upper hinges (3 and 7 amino acids) and were relatively rigid. A long upper hinge will allow the angle between the Fab arms to vary widely, whereas a short upper hinge allows for little Fab arm motion. This hinge flexibility allows divalent recognition of variably spaced antigenic determinants. The ability of Ig molecules to change their conformation from Y to T shape (Nezlin, 1990), greatly facilitates the capacity of antibodies to crosslink antigens. Segmental flexibility has been correlated with complement activation of IgG. Of the nine different IgG molecules studied by Dangl et al. (1988) the flexible antibodies were able to fix complement most effectively. To study the proposed association between hinge length, flexibility and effector function, we made mutants of mouse/human chime& IgG3 antibodies varying the length of the upper hinge and the core hinge, while the rest of the molecule was kept unaltered (Sandlie et al., 1989). Thus, the effect of manipulating the hinge could be studied independently of any effects due to isotype differences in CHl, CH2 and CH3. The antibodies have specificity for the NIP hapten. Five different deletion mutants were constructed as shown in Fig. 1, deleting one or more hinge exons. The resulting antibodies have hinges of 47, 45, 32, 15 and 0 amino acids respectively. The length of the upper hinges of mutant M47 and M32 are identical to that of IgG3 wild type, namely 12 amino acids, whereas the upper hinge of M45 and M15, where the first exon is deleted, consists of 4 amino acids. M47, M45, M32 and Ml5 all reacted with IgG3 hinge-specific antibodies, thus the hinge epitopes were mainly retained. The new CHI H
hl
h2 h3 I4
e$ b ML7 ML5 _M32 Ml5 MO
6
CH2
CH3 1
b
5
l&Q
Fig. 1. Restriction map of the y3 gene. Exons are shown as boxes. H, Hind III; Bg, Bgl II; P, Pst I; S, Sph I. Lines indicate
deletions
in the gene.
hinge
Lower hinge 238 APELLGGP APPVAGP APELLGGP APELLGGP APEFLGGP VPSEVS APNLLGGP
antibodies were tested for their ability to activate complement. M45 and Ml5 activated human complement equally well or better than wildtype IgG3 (Sandlie et al., 1989; Michaelsen et al., 1990~). Both M45 and M15, having upper hinge of four amino acids, activated complement. M45 was equally efficient as wild type IgG3, while Ml5 was more efficient. Therefore, neither a long upper hinge nor a long total hinge is necessary for complement activation. It seems that the long hinge of IgG3 rather than enhancing complement activation by this subclass, downregulates its activation potential. Tan et al. (1990) made similar IgG3 mutants with specificity for the DNS hapten. Segmental flexibility of the antibodies were measured, and found to be low for the mutant with the first hinge exon deleted. These rigid antibodies were also shown to activate complement efficiently. Thus, complement activation does not depend on a high degree of segmental flexibility. However, in several species there is a correlation between segmental flexibility of naturally occurring antibodies and ability to fix complement. Possibly, the organisms have an advantage when the molecules most active in complement activation are able to do extensive Fab-arm waving, and thereby bind variably spaced epitopes, while the nonlytic molecules are limited in this respect.
THE AMINO ACID SEQUENCE OF THE HINGE MODULATES THE ABILITY OF I& TO ACTIVATE COMPLEMENT
IgG4 is completely inactive in complement activation. It is a rigid molecule with a short upper hinge, its core hinge contains the central common hinge sequence (cysteine-proline-proline-cysteine), and the lower hinge is identical to that of IgGl and IgG3 except for a phenylalanine substituted for leucine in position 234. To determine whether the amino acid sequence of the genetic IgG4 hinge is responsible for its lack of effector function, we mutated the hinge exon of Ml5 by in vitro mutagenesis to become identical to that of IgG4. Instead of losing effector function, this IgG3-derived molecule (M15C, Fig. 2) activated complement even better than IgG3 Ml5 (Fig. 3) (Sandlie et al., submitted). Therefore, the amino acid sequence of the IgG4 hinge alone can not be the reason why IgG4 does not activate complement. Chimeric anti-DNS IgG3 molecules with the hinge of IgG4 were also made by Tan et al. (1990) as well
1363
Complement activation and complement mediated lysis Iw3wt IHJ-I-HI
MIS
s
M15C
F
MlSD
s
M15E
B
M15F
B
M15G
0
IgG3
-
IgGG
Fig. 2. Schematic drawing of the IgG3 derived antibody genes. Exons are shown as boxes. as IgG4 molecules with the hinge of IgG3. Functional studies performed on these antibodies also demonstrate that the primary structure of the IgG4 hinge is not the reason why IgG4 does not activate complement. However, in this work the IgG3 mutant with an IgG4 hinge was secreted as a mixture of H2L, molecules and HL half-molecules due to lack of appropriate disulfide bridging, and complement activation could not be measured accurately. Schneider et al. (1988) measured the segmental flexibility of genetically engineered mouse IgGl/IgG2 hybrids with specificity for DNS. It was demonstrated that proper “matching” of the iv-terminal part of the CHl domain and the hinge was important for the flexibility of the IgG molecule. The
N-terminal part contains a loop formed by residues 131-139 in CHl (EU numbering). It has previously been demonstrated by X-ray crystallography of human IgGl Kol that this loop is very near to the hinge (Marquart et al., 1980). The only differences between IgG4 and IgG3 in the N-terminal part of CHl are in residues 137 and 138 (Fig. 4). We mutated these two residues in CHl of IgG3 to become identical to those of IgG4 (M15D, Fig. 2). Complement activation was measured and found to be equal to that of wild-type IgG3 (Fig. 3) suggesting that of the mutations tested, those affecting the hinge are the most important in modulating this effector function. Segmental flexibility of the molecules have not been studied. When this mutation was introduced into the IgG3 molecule with the hinge of IgG4 (Ml5E, Fig. 2), complement activation improved relative to M 15D (Fig. 3). We also made molecules which are IgG4-like in CHl and the hinge, and IgG3-like in CH2 and CH3 (Ml5G, Fig. 2). These molecules activate complement as efficiently as Ml5 These results are unexpected, since Fc fragments from human IgG4 have been shown to bind Clq with an affinity comparable to that of the corresponding fragment from IgGl (Isenmann et al., 1975). The results from Isenmann et al. (1975) were interpreted to suggest that IgG4 Fab may prevent Clq binding by blocking the access to the IgG4 Fc Clq binding site. We have shown that Fab-hinge from IgG4 positively modulates Clq binding on IgG3. It remains to be shown whether structural features in Fc of IgG4 alone is responsible for the lack of Clq binding observed for IgG4, or whether an interplay between Fab and Fc is involved.
Ch
Absorbance
1.8 , t 1.6
1
1.4
t
+k
Ml5
--A--
lgG3
-a-
M15C
-A-
M15D
-t,-
M15E
+
M15F
-
M15G
wt
t 1.2
1
t
’ t 0.8 t 0.6 0.4 0.2 0 3.7
11.1
33.3
100
300
900
Concentration(ng/ml)
Fig. 3. EIA analysis of complement activation. The experiments were performed in microtiter wells coated with NIP-BSA conjugate. Serial dilutions of chimeric antibodies were added to the wells. Normal fresh serum diluted 1: 30 in isotonic Verona1 buffer was used as complement source, and complement activation measured by the binding of rabbit anti-Clq antibodies to the wells. MlMM
28,12-0
INGER SANDLIE and
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TERJEE. MICHAELSEN
CML
Concentratii
MPG.-FOb'
I
of chime& antibodes ng/ml
Fig. 6. Complement mediated lysis of hinge mutants of IgG3. The molecules tested were wild type IgG3 (17-IS-1%15), a variant with 47 amino acid hinge (17-1515), a variant with 32 amino acid hinge (17-15), a variant with a 15 amino acid hinge (1 S), a variant with 45 amino acid hinge (15-15-15) and a variant without hinge (0). IgGl is also included for comparison.
THE CHZ DOMAIN CARRIES FEATURES CRITICAL FOR COMPLEMENT ACTIVATION IN ADDITION TO THE Clq BINDING MOTIF
Clackson and Winter (1989) have created hybrids of lytic and non-lytic murine antibodies with specificity for the NIP hapten, and have shown that the CH2 exon of the IgG2b antibody active in complement-mediated lysis can confer lytic ability when replacing the CH2 exon on the usually inactive IgGl. This also argues against the hypothesis that the length of the upper hinge determines the ability of an antibody to activate complement. The lower hinge of mouse IgGl is rather short, and lacks the three amino acids usually found in positions 233, 234 and 236. Human IgG4 also has an abnormality in the lower hinge, namely the phenylalanine in position 234. The IgG subclasses have evolved independently in the two species, mouse and man, and IgG4 is not the human equivalent to mouse IgGl (Callard and Turner, 1990). Mouse IgGl can be shown to display a slight Clq binding ability at some experimental conditions, while IgG4 totally lacks complement activation ability regardless of conditions (Michaelsen et al., 19906). Therefore the structures rendering these two antibodies non-lytic may be different. Comparing the amino acid sequence of IgG3 and IgG4 in Fc, six amino acids differ in CH2 and three in CH3, not counting the residues that are unique to IgG3, and where IgGl, IgG2 and IgG4 use the same residue. In CH2, these are all located to the upper half of the globular domain structure, and several are located in hinge proximal bends formed between p-strands (Fig. 5) (Deisenhofer, 1981). This area of the globular CH2 domain may prove to be of major importance for Clq binding and complement activation. The carbohydrate bound to Asn 297 in CH2 is necessary for Clq-binding (Duncan and Winter, 1988) as aglycosylated antibodies produced by an Asn/Ala amino acid substitution at residue 297 show a three-fold lower association constant for Clq and
does not activate C 1. Studies by Lund et al. (in press) suggest that aglycosylation results in very localized structural changes in the lower hinge. It is suggested that Clq interacts both with the “core” binding motif and the lower hinge, and that aglycosylation results in loss of accessibility to the hinge. Tight Clq binding is however not sufficient for complement activation and complement lysis to occur, since both wild type antibodies and mutant variants possessing Clq binding activity can be non-lytic (Duncan and Winter, 1988; Bindon et al., 1988).
COMPLEMENT
MEDIATED
LYSIS
When comparing the four human IgGs in complement mediated lysis (CML), we found that IgG3 was the most efficient in complement activation, as well as lysis (Michaelsen et al., 1990b; Garred et al., 1989). To measure CML, SRBC was incubated with Na “0, and sensitized with the hapten NIP antigen as follows: NIP was conjugated to rabbit anti-SRBC Fab as described (Michaelsen et al., 1990a). Both the amount of Fab fragment that was bound to a total of 10’ SRBC and the number of hapten molecules that were conjugated to each Fab fragment was varied. After incubation at 37°C the cells were washed and diluted to 2-3 x 10’ cells/ml, Serial dilutions of chimeric anti-NIP antibodies were added to the target cell solution before human serum was added. After a 30-min incubation at 37°C the amount of released “Cr was measured with a gamma counter. The activity of the different subclasses could thus be measured varying the epitope concentration and also the epitope density, or “patchiness” on the target cells. It was indeed found that the activity of the different subclasses do depend on these features. IgGl, however active at high epitope concentration and epitope patchiness, was found to have a low activity at low epitope concentration and low epitope patchiness (Fig. 6). IgG3 was the most active at a wide range of conditions tested. It is noteworthy that the short hinge mutant Ml5 proved more lytic than
Complement activation and complement mediated lysis
Fig. 4. Ribbon through the CHl domain from human IgGl (Marquart et al., 1980). Residues which differ between IgG3 and IgG4 in a hinge proximal loop are indicated with red balls. The heavy chain is yellow, the light chain is green. The start of the hinge is localized by an arrow.
1365
1366
INGER
SANDLIEand TERJEE. MICHAEUEN
Fig. 5. a-Carbon trace of human Fc (Deisenhofer, 1981). Residues which differ between IgG3 and IgG4 are marked with red balls. The Clq binding site in CH2 is marked with yellow balls.
Complement activation and complement mediated lysis Table 2. Antibody induced complement mediated lysis Antibody tested
Antibody concentration needed for 50% lysis (n&l)
IgG3 wild type Ml5 M15C
36 4 0.6
The target cells (SRBC) were sensitized with NIP coupled to Fab anti-SRBC. The hapten: Fab ratio was 15:1, and 400 ng hapten was added to lo8 target cells.
IgG3 wildtype (Michaelsen et al., 1990a), and that the mutant M15C was even more lytic (Table 2). Work by others employing the same chimeric mouse/human antibodies with antigen specificity for NIP (Bruggemann et al., 1987; Bindon et al., 1988) suggest that IgGl is superior in CML due to higher activity at a later stage in the complement cascade, namely C4 activation. Our work does not confirm these findings, and the discrepancy probably reflects the differences in the in vitro experimental conditions employed. Experiments in vivo will eventually determine how efficient the various antibodies are in lysing target cells
CONCLUSIONS
Recent work with genetically engineered monoclonal antibodies show that the hinge region modulates complement activation. However, neither a long upper hinge nor high segmental flexibility is necessary for efficient complement activation. Also, structures in the lower hinge or hinge proximal residues in the globular CH2 domain seem to be important. Acknowledgements-The help of Dr Christian Ramming to create the computer graphics is greatly appreciated. The work was supported by grants from The Norwegian Cancer Society.
REFERENCES
C. I., Hale G., Bruggemann M. and Waldmann H. (1988) Human monoclonal IgG isotypes differ in comp-
Bindon
lement activating function at the level of C4 as well as Clq. J. exp. Med. 168, 1277142. Bindon C. I., Hale G. and Waldman H. (1990) Complement activation by immunoglobulin does not depend solely on Clq binding. Eur. J. Immun. 20, 277-281. Bruggemann M., Williams G. T., Bindon C. I., Clark M. R., Walker M. R., Jefferis R., Waldman H. and Neuberger M. S. (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J. exp. Med. 166, 1351-1361. Burton D. R. (1987) Structure and function of antibodies. In Molecular Genetics of Immunoglobulin (Edited by Calabi F. and Neuberger M. S.), p. 1. Elsevier, Amsterdam. Callard R. E. and Turner M. W. (1990) Cytokines and Ig switching: evolutionary divergence between mice and humans. Zmmun. Today 11, 200-203. Clackson T. and Winter G. (1989) ‘Sticky feet”-directed mutagenesis and its application to swapping antibody domains. Nucl. Acids Res. 17, 10163-10170. Dangl J. L., Wensel T. G., Morrison S. L., Stryer L., Herzenberg L. A. and Oi V. T. (1988) Segmental flexibility and complement fixation of genetically engineered
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chimeric human, rabbit and mouse antibodies. EMBO J. 7, 1989-1994. Deisenhofer J. (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9 and 2.8 8, resolution. Biochemistry 20, 2361-2370. Duncan A. R. and Winter G. (1988) The binding site for Clq on IgG. Nature 332, 738-740. Endo S. and Arata Y. (1985) Proton nuclear magnetic resonance study of human IgGl and their proteolytic fragments: structure of the hinge region and effects of a hinge region deletion on internal flexibility. Biochemistry 24, 156lLl568. Garred P., Michaelsen T. E. and Aase A. (1989) The IgG subclass pattern of complement activation depends on epitope density, antibodyand complement concentration. Stand J. Immun. 30, 379-382. Huber R., Deisenhofer J., Colman P. M., Masaak M. and Palm W. (1976) Crystallographic structure studies of an IgG molecule and a Fc fragment. Nature 264, 415-420. Isenmann D. E.. Dorrineton K. J. and Painter R. H. (1975) The structure’and function of immunoglobulin do&&n: II. The importance of interchain disulfide bonds and the possible role of molecular flexibility in the interaction between immunoglobulin and complement. J. Immun. 114, 172661729. Ito W. and Arata Y. (1985) Proton nuclear magnetic study of the dynamics of the conformation of the hinge segment of human Gl immunoglobulin. Biochemistry 24, 6467-6474. Johnson P. M., Michaelsen T. E. and Scopes P. M. (1975) Conformation of the hinge and various fragments of human IgG3. &and. J. Immun. 4, 113-119. Lund J., Tanaka T., Takahashi N., Sarmay G., Araka Y. and Jefferis R. (1990) A protein structural change in aglycosylated IgG3 correlates with loss of human FcRI and human FcRIII binding and/or activation. Molec. Immun. (in press). Marquart M., Deisenhofer J., Huber R. and Palm W. (1980) Crystallographic refinement and atomic models of the _.._ - _ intact immunoglobulin molecule Kol and its antigenbinding fragment at 3.0 8, and 1.9 8, resolution. J. molec. Biol. 141, 3699391. Michaelsen T. E. (1976) Indications that the CH2 homology region is not a regular domain. Stand. J. Immun. 5, 1123-I 128. Michaelsen T. E., Aase A., Westby C. and Sandlie I. (1990a) Enhancement of complement activation and cytolysis of human IgG3 by deletion of hinge exons. &and. J. Immun. 32, 517-528. Michaelsen T. E., Garred P. and Aase A. (199Ob) Human IgG subclasses induce complement-mediated cytolysis differently upon changes in antibody affinity, complement concentration and epitope density. Eur. J. Immun. (in press). Nezlin R. (1990) Internal movements in immunoglobulin molecules. Adu. Immun. 48, l-39. Poon P. M., Schumaker V. N., Phillips M. L. and Strang C. J. (1983) Conformational and restricted segmental flexibility of Cl, the first component of human complement. J. molec. Biol. 168, 563-577. Reid K. E. M. and Porter R. R. (1981) The proteolytic activation systems of complement. A. Rev. Biochem. 50, 433-464. Sandlie I., Aase A., Westby C. and Michaelsen T. E. (1989) Clq binding to chimeric monoclonal IgG3 antibodies consisting of mouse variable regions and human constant regions with shortened hinge containing 15 to 47 amino acids. Eur. J. Immun. 19, 1599-1603. Sandlie I., Norderhaug L., Brekke 0. H. A., Bremnes B., Sandin R., Aase A. and Michaelsen T. E. The amino acid sequence of the hinge modulates the lytic ability of IgG. Submitted.
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Schneider W. P., Wensel T. G., Stryer L. and Oi V. T. (1988) Genetically engineered immunoglobulins reveal structural features controlling segmental flexibility. Proc. natn.
Acad. Sci. U.S.A. 85, 2509-2513.
Tan L. K., Shopes R. J., Oi V. T. and Morrison S. L. (1990) Influence of the hinge region on complement activation, Clq binding, and segmental flexibility in chimeric human immunoglobulins. Proc. natn. Acad. Sci. U.S.A. 87,
162-166.