Structural Features of Human Immunoglobulin G That Determine Isotype-Specitic Differences in Complement Activation by Mi-Hua Tao,* Richard I.F

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Structural Features of Human Immunoglobulin G that Determine Isotype-specitic Differences in Complement Activation By Mi-Hua Tao,* Richard I.F. Smith,* and Sherie L. Morrison*r

From the "Department of Microbiology and Molecular Genetics and IThe Molecular Biology Institute, University of California, Los Angeles, California 90024

Summary Although very similar in sequence, the four subclasses of human immunoglobulin G (IgG) differ markedly in their ability to activate complement. Glu318-Lys320-Lys322 has been identified as Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 a key binding motif for the first component of complement, Clq, and is present in all isotypes of Ig capable of activating complement. This motif, however, is present in all subclasses of human IgG, including those that show little (IgG2) or even no (IgG4) complement activity. Using point mutants of chimeric antibodies, we have identified specificresidues responsible for the differing ability of the IgG subclasses to fix complement. In particular, we show that Ser at position 331 in 3/4 is critical for determining the inability of that isotype to bind Clq and activate complement. Additionally, we provide further evidence that levels of Clq binding do not necessarily correlate with levels of complement activity, and that Clq binding alone is not sufficient for complement activation.

he classical pathway of complement activation is initi- tivation ability and an IgG4 with the hinge of IgG3, although T ated by immune complexes composed of antigen and ei- as flexible as wild-type IgG3, displays no detectable comple- ther IgM or IgG Abs. Although the C region domains of ment activity. It thus appears that it is not the short, inflex- all four human IgG subclasses share virtually identical amino ible hinge but other structural features located in the H chain acid sequences, they differ markedly in their ability to acti- that lead to the inactivity of IgG4. vate complement. Studies of mouse-human chimeric Abs have C.2 has been implicated as the domain responsible for iso- shown that IgG3 and IgG1 are effective in activating com- typic differences in complement fixation. Earlier Ab fragmen- plement, IgG2 fixes complement poorly, and IgG4 appears tation studies (8-10) suggested a crucial role for C.2 in ac- completely defcient in the ability to activate complement (1, tivating complement by showing that Fab (IgG depleted of 2). The structural bases for these isotype-specific differences CH3) and C.2 fragments bind C1 and activate complement, in complement activation have not been defined and are the whereas Fab and C.3 fragments do not show any activity. subject of this investigation. Moreover, aglycosylated IgG (lacking carbohydrate side chains The hinge region, which separates the Fab from the Fc, in C.2) is unable to activate complement (1I, 12). Domain- has been implicated in determining these isotypic differences shuffled chimeric Abs provide further direct evidence that in part because the sequences of the hinge regions, in con- the isotypic variations in complement activity are determined trast to the other C region domains of the 3' isotypes, are by this domain. IgG2 with the C.2 domain from IgG3 per- remarkably diverse (2, 3) and also because two hinge-deleted forms complement-mediated cell lysis almost as well as wild- human IgG1 myeloma proteins lost both the ability to acti- type IgG3, and the replacement of C.2 in IgG3 with the vate complement and to bind its first component, C1 (4). corresponding domain from IgG2 decreases its capacity to Additionally, hinge length and Fab arm segmental flexibility fix complement to a level comparable with that of wild-type of IgG Abs from different species were found to correlate IgG2 (13). By contrast, the exchange of C.1 or C.3 domains with the ability to activate complement (2, 5). Recently, how- has no effect on complement activation (7, 13). Studies of ever, we and others (6, 7) using genetically engineered Abs domain switch Abs of murine IgG subclasses also confirmed have shown that whereas a hinge region is essential for Clq the importance of C.2 in determining the isotypic differ- binding and complement activation, the hinge does not ap- ences (14). We further localized the structural features leading pear to determine isotypic differences in these functions, and to the inactivity of human IgG4 to the COOH-terminal part there is not an absolute correlation between segmental flexi- of C.2, from residues 292 to 340 (13). bility and the ability to activate complement. An IgG3 with In the present study we identify specificpolymorphic amino the rigid hinge of IgG4 exhibits significant complement ac- acids in the C.2 domain of human IgG that account for sub-

661 J. Exp. Med. @ The RockefellerUniversity Press 0022-1007/93/08/0661/05 $2.00 Volume 178 August 1993 661-667 class differences in complement activation. Mutations intro- trifugation of the plate, and 50/~1 of supernatant counted in a gamma duced into chimeric IgG1, IgG3, or IgG4 show that the pres- counter. Each point was assayedin triplicate and the percent lysis ence of Ser instead of Pro at residue 331 in wild-type IgG4 calculated. For Abs that contained a mutation that restored activity, contributes significantly to its inability to bind Clq and acti- multiple protein preparations were analyzed. vate complement. In addition, we provide evidence that sub- C1q Binding. All steps were carried out in HBSS plus 0.02% sodium azide and plates were washed six times between each step stitutions at residues 276 and 291 can influence the ability with the same buffer. 96-weU plates (Immulon-2; Dynatech Labora- to activate complement, although apparently not by affecting tories Inc., Chantilly, VA) were coated with 100 #l/well DNS- Clq binding, suggesting the involvement of additional Ab- BSA (40:1 substitution ratio, 10/~g/ml, overnight at 4~ then dependent steps in the classical pathway of complement ac- blocked with 200/~l/well 3% BSA (overnight at 4~ All Abs tivation. Other polymorphic residues in C.2 are also dis- were diluted in 1% BSA, applied in a volume of 100/~l/well, and cussed. incubated at room temperature for 2 h or overnight at 4~ All assays were carried out in quadruplicate. First, anti-DNS Abs were Materials and Methods bound at a concentration of 20 #g/ml. The plates were washed and normal human serum diluted to 0.25% was applied in a volume In Vitro Mutagenesis and Construction of Chimeric IgG Mole- of 100 #1 at 37~ for 2 h. The detecting Ab was goat anti-human cules. The expressed Vx and VH genes from the mouse anti- Clq (Atlantic Antibodies, Stillwater, MN) diluted 1:10,000, and dansyl (DNS) 1 hybridoma 27-44 were joined to human CK in the the developing agent was swine anti-goat IgG-alkaline phospha- pSV2AHneo expression vector and to human IgG H chain in the tase conjugate (Boehringer Mannheim, Indianapolis, IN) diluted Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 pSV2AHgpt vector, respectively (15). The human genomic ~/C 1:20,000. 100/~1 p-nitrophenyl phosphate (Sigma Chemical Co.) region genes were cloned as SalI/BamHI cassettes. This makes it at 0.5 mg/ml in diethanolamine was added as phosphatase sub- convenient to shuffle the genes between different ptasmids and to strate, and the OD read at 410 nm. join different H chain genes to the same V.. The SalI/BamHI cassettes containing human C3' genes were cloned into the polylinker region of M13mp18 or M13mp19 for Results mutagenesis. To construct the intra-C,2 domain switch variants Our previous domain switch experiments clearly demon- of IgG1 and IgG4, the cloned IgG genes were digested with Sail and SaclI (which cleaves within C.2) and the SalI/SacII fragments strated that the CH2 domain of IgG contains sites important were reciprocallyexchanged between 3/1 and 3~4. Oligonucleotide- for interacting with Clq and initiating activation of the com- mediated site-directed mutagenesis was done based on the two- plement cascade (13). Human IgG1, IgG3, and IgG4 exhibit primer method of Zoller and Smith (16) with modifications and very few polymorphisms in their C~2 domains (Table 1), but the mutations confirmed by sequencing. differ profoundly in their ability to activate complement by Production of Transfectoma Proteins. P3X63Ag8.653, an Ig non- the classical pathway. We set out to establish which amino producing mouse mydoma cell line, was transfectedsimultaneously acid polyporphisms determine the isotype-specific differences with the H and L chain expression vectors by dectroporation (17). in complement activation. To address this question, a set of Transfectants were selected with G418 (Gibco, Grand Island, NY) at 1.0 ms/m1, and surviving clones were screened for Ab production by ELISAusing DNS/BSA-coatedplates. The amount of bound Table 1. Polymorphisms in C,2 among IgG1, IgG3, chimeric Ab was determined using alkalinephosphatase-conjugated and lgG4 polyclonal goat Ab (Sigma Chemical Co., St. Louis, MO) against human IgG C regions. Clones producing large quantities of anti- Residue IgG1 IgG3 IgG4 DNS Ab were expanded and maintained in IMDM containing 5% calf serum. Chimeric Abs were purified by DNS-coupled affinity 234* Leu* Leu Phe chromatography as described previously (12). The concentrations of purified Abs were determined by a bicinchoninic acid-based pro- 268 His His Gln tein assay (BCA; Pierce, Rockford, IL). 274 Lys Gln Gln Complement-mediatedHemolysis. SILBCwere coated with DNS- 276 Asn Lys Asn BSA (0.25 mg/ml DNS-BSA, 5% SR.BC, in 150 mM NaC1, 0.25 291 Pro Leu Pro mM CrC13, pH 7.0, for 1 h at 30~ and loaded with SlCr- sodium chromate (Amersham Corp., Arlington Heights, IL). The free SlCr-sodinm chromate was removedby washing the cells three 296 Tyr Tyr Phe times in 10 ml of fresh geI-HBSS buffer (0.01 M Hepes, 0.15 M 300 Tyr Phe Tyr NaC1, 0.5 mM MgCI2, 0.15 mM CaC12, and 0.1% gelatin, pH 327 Ala Ala Gly 7.4). Chimeric Abs in geI-HBSS at various concentrations were 330 Ala Ala Ser added to round-bottomed, 96-weU plates (Corning Glass Works, Coming, NY) in a volume of 50/~1. Then 50 #1 of 2% SlCr- 331 Pro Pro Ser loaded SRBC and 10 times the amount of guinea pig complement (Colorado Serum Co., Denver, CO) required to produce lysis of * Residue number is based on the EU numbering system. 50% of the cells, preabsorbed against unlabeled SR.SC, in a volume *Sequences of IgG subclasses are taken from Kabat et al. (3) and of 25/~1 were added to each well sequentially. The plates were in- confirmed by sequence analysis of the H chain genes used in these ex- cubated at 370C for 45 min, unlysed StLBC were pelleted by cen- periments. (-) Position of C.2 exchange in IgG (1/4) and IgG (4/1) hybrid 1 Abbreviations used in this paper: DNS, dansyl. proteins.

662 HumanIgG Isotype-specificDifferences in Complement Activation Table 2. Nomenclatureand Constructs of C Region Domains for Chimeric Abs Used in this Study IgG3(Asn276) I ~" xsGI I Region E.xons '.oI'L,.2'~ I / Name CHI H Cta2 Crt3 Description 60,.I IgG7

IgG 1 I Wild type lgG 1 .~ IgGl(Lys276) I T I IgGI Asn276--)Lys "~ 40- lgG I(Leu29X) | T lgGl Pro291--->Leu IgG l(Ser330) II T lgGl Ala330-->Ser 20- IgG l(Ser331) II ~ lgGl Pro33 l--+Ser IgG3 Wild type lgG3 0 ...... i IgG3(Asn276) ..... ms"" ~- -- ~ IgO3 Lys276--->Asn .1 1 10 100 IgG3(Ser331) ~ ~. --..--~_'-r IgG3 Pro331-/,Ser Chimeric IgG (Bg/ml) lgG4 -'1 I-0"t I"1 I" Wild type IgG4 Figure 1. Complement-mediatedhemolysis by wild-typeand pointmu- IgG4(Tyr296) "1 ~ I-'I t" IgG4 Phe296-~Tyr tants of IgG1 and lgG3. stCr-loaded, DNS-BSA-coated SRBC were in- Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 lgG4(Ala330) -I ~ ~M I- IgG4 Ser330---)Ala cubated with varying concentrations of Ab in the presence of guinea pig complement and percent cell lysis calculated. IgG3 mutated to resemble IgG4(Pro331) "-I ~ ~l-I '" ]- IgG4 Ser331--~Pro lgG1 at position 276 shows an activity reduced approximately to the level IgG(4/X) -'1 ~ 4,4,4/1-1 hybrid of wild-type IgG1. IgG3-1ike mutants of IgG1 however, do not show in- lgG(l/4) ~ I" I D, 1/4,,4 hybrid creased complement activity, rather they too are impaired relative to wild type IgG 1/4 (Pro331) ~ ,1"- IgG(l/4) Ser331---)Pro

(~2) Location of mutated amino adds. Abs to carry out complement-mediated hemolysis and their ability to bind Clq. However, it should be noted that whereas guinea pig complement was used in the direct lysis assay, mouse-human chimeric Abs having identical antigen-com- human complement was used to measure Clq binding. IgG3 bining sites (anti-DNS) and L chains but with different H was consistently more effective than IgG1 in complement- chain C region domains were generated using previously de- mediated hemolysis (Fig. 1), but both isotypes were equiva- scribed techniques (15, 18). The point mutations were made lent in their ability to bind Clq (Fig. 2). The result obtained in the context of either wild-type human 3/1, 3'3, 3,4, or a in the current study differs slightly from our previously ob- C region in which yl and 3'4 were shuffled at amino acid servation that IgG3 was seen to bind Clq better than IgG1 292, as described previously (13). The names of the mutant (12). However, a different version of the Clq binding assay proteins and the amino acids mutated in their C.2 domains was used in the earlier study. It is interesting that whereas are shown in Table 2. All chimeric Abs were purified from exchanging residues at position 276 in IgG1 and IgG3 dearly culture supernatants using DNS isomer affinity chromatog- affects both molecules negatively in overall complement ac- raphy (12) and used in the complement-mediated hemolysis tivation, neither mutation significantly affects Clq binding and Clq binding assays. (Fig. 2). IgGl(Leu291), also binds Clq as welt as wild type, Analysis of Residues Polymorehic between IgGI and lgG3. In but is impaired in the direct lysis assay. previous studies we have consistently found anti-DNS IgG3 Role of Pro331. IgG4 is unable to activate complement to be about 10 times more effective than IgG1 in comple- or bind Clq, a deficiency that can be localized to the COOH ment consumption and complement-mediated cell lysis (2, terminus of C.2 (13). These studies had shown that the hy- 12). Within the C,2 domain, human IgG1 and IgG3 differ brid protein IgG1/4, in which the C region switches from from each other at only four residues: 274 (Lys vs. Gln), 276 IgG1 to IgG4 at amino acid 292, is unable to activate com- (Asn vs. Lys), 291 (Pro vs. Leu), and 300 (Tyr vs. Phe) (Table plement whereas IgG4/1, the molecule with the reciprocal 1). We focused our attention on the polymorphisms at residues exchange, can, albeit not as well as IgG1. Within C.2, the 276 and 291. Indeed, we find that substitution of Lys276 with IgG1/4 protein differs from IgG1 at four residues: Phe296 Asn in IgG3 (IgG3[Asn276]) reduces its ability to activate instead of Tyr, Gly327 instead of Ala, Set330 instead of Ala, complement to a level comparable with that seen in IgG1 and Ser331 instead of Pro. The Set/Pro polymorphism at res- (Fig. 1). However, the reciprocal mutation, Asn 276---Lys idue 331 seemed to be a good candidate for contributing to in IgG1 (IgG1[Lys276]), does not improve its ability to acti- the differences in activity, both because of the nature of the vate complement but instead results in a protein with im- substitution and because of the position of Pro exposed on paired ability. Therefore, whereas the Lys 276+Asn substi- the surface of the protein (Fig. 3). Indeed, we find that sub- tution can make IgG3 functionally similar to IgG1 in its ability stitution of Pro at position 331 in IgG4 (IgG4[Pro331]) results to activate complement, none of the single amino acid changes in a molecule that can now bind Clq and mediate complement- tested suffice to improve IgG1 to the level of IgG3. directed lysis (Figs. 2 and 4 A). When this substitution is There was a lack of correlation between the ability of the made in the context of the IgG1/4 protein, the resulting mol-

663 Tao et al. IgGl IgG4/l IgG1(Lys276) lgG3(Asn276) lgGl(Le u291) I ::::::%, I gG l(Se r330) / IgG3

IgGl(Se r331) ~//////I///////2q IgG3(Ser331) ~~///~'-'q Effect of Mutation on Clq Binding IgG1/4(Pro331) none IgG4(Pro331) [] negative

IgG4(Tyr296) 9 positive

IgC4(Ser330) _q standard Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 deviation IgG1/4 Figure 3. Three-dimensional structure of human IgG1 Fc. Amino acids |gG4 that are discussed in the text are darkened and indicated by arrows. Ala327 1 1 1 t l I is buried within the molecule. For clarity, one H chain is shaded slightly 20 40 60 80 100 120 more than the other and associated carbohydrates are shaded like the op- posing H chain. The graphic is based on the coordinates from Diesenhofer Percent of IgG1 Binding (%) (23) and was produced using the program Maclmdad (Molecular Applica- tions Group, Stanford University, Stanford, CA). Figure 2. Clq binding by wild-type and point mutants of IgG1, IgG3, IgG4, and the 1/4 hybrid proteins. Clq bound to Ag-Ab complexes on binding ability of the molecule (Figs. 2 and 5). When the a microtiter dish was detected with goat anti-Clq Ab and swine anti-goat reciprocal substitution was made in IgG4 (IgG4[Ala330]) the IgG conjugated to alkaline phosphatase. Substitution of Pro for Set at position 331 in IgG4 and the IgG1/4 hybrid results in Abs now capable of Ab remained unable to activate complement or bind Clq. binding Clq (ll). The reciprocalmutation in IgG1 and IgG3 greatly reduces Similarly, substitution of the polymorphic residue at posi- their ability to bind Clq although only to the level of the Pro331 mutants tion 296 (IgG4[Tyr296]) left the molecule deficient in its ability of IgG4 and IgG1/4 ([]). Our mutations at positions 276, 291, 296, and to activate complement. Thus, we find no evidence that resi- 330 in IgG1, IgG3, and IgG4 showed little or no affect on Clq binding dues 296 or 330 determine isotype-specific differences in com- (D). Each protein was tested in quadruplicate and in two or more inde- pendent assays. Error bars represent the variation between assays. plement activation. ecule, IgG1/4(Pro331), also binds Clq and is even more Discussion efficient in complement-mediated lysis than IgG4(Pro331). Activation of complement through the classical pathway To further prove that Pro331 is critical for complement acti- is initiated by the interaction of Clq and IgG or IgM in an vation, we created Ser331 mutants of both IgG1 and IgG3. immune complex. Previous studies (8-10) have indicated that The substitution of Ser for Pro at position 331 in IgG1 the C.2 domain of the H chain of IgG contains structures (IgGl[Ser331]) results in complete loss of the ability to carry critical for complement activation and that complement- out complement-mediated hemolysis (Fig. 4 B). It is in- activating capacities can be transferred between active and teresting, however, that the molecule binds Clq as well as inactive IgG subclasses with Ca2, whereas the exchange of IgG1/4(Pro 331) (Fig. 2), which is quite effective in carrying C.1, hinge, or Ca3 produces little or no effect (7, 13, 14). out complement-mediatedhemolysis (Fig. 4). The same sub- Three possible C1 binding sites have been proposed: His285- stitution in IgG3 (IgG3[Ser331]) also profoundly impairs, but Arg292 (19), the residues on the last two antiparatld fl strands does not abolish, its ability to carry out complement-mediated (20), and Lys290-Glu295 (21). Based on protein engineering hemolysis, and reduces its Clq binding to the intermediate studies of mouse IgG2b, three charged amino acids (Glu318, level characteristic of IgG1/4(Pro331), IgG4(Pro331), and Lys320, and Lys322) located on one fl strand of C.2 were IgGl(Ser331). proposed as constituting the essential Clq binding motif (22). OtherPolymorphic Residues in IgG1 and IgG4. Although However, this core binding motif is conserved in all four the replacement of Set 331 with Pro in IgG4 partially re- human IgG subclasses that exhibit dramatic differencesin com- stores its activity, IgG4(Pro331) is still far less ef~dent than plement activation, including IgG4, which is completely in- wild-type IgG1 in activating complement. Thus, additional active and does not bind Clq. Clearly, additional structural mutant proteins were made to investigate the role of other elements must determine the isotype-specific differences in residues polymorphic between the two isotypes. Substitu- IgG-complement interaction. We have now focused our at- tion of Set for Ala are residue 330 in IgG1 (IgGl[Ser330]) tention on the polymorphic amino acids located in Ca2 did not significantly alter the complement activating or Clq which may determine these differences.

664 Human IgG Isotype-specific Differences in Complement Activation 60. A 8o IgOI igOl IgG4/I 1~31(Ser330) IgGI/4(Pro331) 50' nxgO4 6O A~ IgG4(Pro331) i.--t A IIG4(AIa330) z~1/4 ~v"- - 0 Ig~ 40"

r~ 30"

20" ~ 2 10'

1 1 10 100 0 el ...... ,'I . . .i,i..... tl ...... I 1 I0 I00 Chimeric IgG (p.g/ml) B i00, IgG3 O IgOl Figure 5. Complement-mediatedhemolysis by wild-tyl~and point mu-

IgG3(S~331) tants of IgG1 and I~o4. Mutating position 330 of I$G1 to resembleIgG4 Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 does not affectthe ability of the moleculeto direct lysis. Similarly,IgG1- like position 296 and 330 mutants of lg~ remain completelyinactive.

~ 40- the importance of the context of the entire domain in determining the contribution of specific residues. Human IgG4 is unable to bind Clq and is incapable of 20- complement activation. It has been postulated that either reduced segmental flexibility (2, 5) or steric hindrance by Fab 0 ...... i arms (24) caused by a short, rigid IgG4 hinge was respon- .1 1 10 100 sible for the inactivity. However, recent Ab engineering ex- Chimeric IgG (I.tg/ml) periments by us and others (6, 8) have provided evidence against Figure 4. Complement-mediatedhemolysis by IgG wild-typeand po- this hypothesis. An IgG4 molecule with the flexible hinge sition 331 mutants. (.4) IgG4 and the IgG1/4 hybrid show no activity. region of IgG3 fails to bind Clq or activate complement, However, substitution of Pro for Set at position 331 results in Abs now indicating that a structural lesion other than the rigid hinge capable of directing lysis. (B) Substitution of Set for Pro at position 331 leads to the inactivity of IgG4. By intra-domain exchange severelyimpairs the abilityof IgG3 to direct]ysis and completelyabolishes IgG1 activity. between IgG1 and IgG4 we have located the structures responsible for the inactivity of IgG4 to the COOH-terminal part of C.2 (from residue 292-340) (13). We now show that residue 331 is a critical amino acid for determining the isotypic IgG3, which is more efficient than IgG1 in complement differences in complement activation. Substitution of Ser331 consumption and complement-mediated cell lysis (2, 12), with Pro in IgG4 or the IgG1/4 hybrid results in Abs now differs in C.2 from IgG1 at only four amino acids (Table 1). capable of binding Clq and of complement-mediated lysis. In the three-dimensional structure (23), residue 276 is in close In addition, the reciprocal exchange of Pro331 to Ser in IgG3 contact with the proposed 318-320-322 Clq binding motif or IgG1 dramatically decreases or completely abolishes the (Fig. 3), and it is conceivable that the charged side chain of capacity to activate complement. Residue 331 lies in a pep- Lys276 in IgG3 contributes to the Clq-IgG interaction. In tide loop between the last two ~/strands of C.2 and is in fact, we found that substitution of Lys276 with Asn in IgG3 close proximity to the 318-320-322 Clq binding motif. It (IgG3[Asn276]) reduces its complement-activating capacity is feasible that Pro331 is either part of the Clq binding site to the level seen in IgG1. However, the reciprocal change in or is required for maintaining the appropriate structures for IgG1 (IgGl[Lys276]) does not increase its activity. Instead, Clq-IgG interaction. Moreover, Pro436 in C/~3 is analogous the mutant Ab is somewhat impaired in its capacity to mediate to Pro331 in C3~2 and a mutant IgM Ab with Pro436-~Ser cell lysis. Furthermore, although both mutations affect com- showed a >50-fold reduction in complement activity (25). plement-mediated cell lysis, neither has a significant effect This observation further supports a critical role for Pro331. on Clq binding. These results suggest that the side chain It is interesting that the Pro331-~Ser mutation in IgG3 also of Lys276 is involved in some subsequent Ab-dependent step caused a 10-fold reduction in its affinity for the human high of the cascade and that in the context of IgG1, it does not affinity Fc3~ receptor (26). Therefore, the presence of Pro at assume a proper configuration for effective complement acti- residue 331 is important for both complement activation and vation. Similarly, mutation at residue 291 in IgG1 from Pro Fc receptor binding. to Leu (as found in IgG3) decreases its ability to activate com- The IgG1 (Ser331) mutant is of particular interest because, plement without affecting Clq binding, again illustrating although it binds Clq, it fails to carry out lysis, demonstrating

665 Tao et al. dearly that Clq binding alone is not sufficient for comple- involvement of residue 274 in complement activation was ment activation. This conclusion is supported by previous thought to be unlikely since a mutation at this residue in results in which a mutant of IgG3 lacking carbohydrate in murine IgG2b did not alter its activity (22). Moreover, the C.2 could bind Clq but not consume complement (12). same amino acid, Gin, is found in both complement-active Our results suggest that amino acids other than Pro331 and IgG3 and complement-inactive IgG4. In light of our results, polymorphic between IgG1 and IgG3 are important for Ab- however, this residue does warrant investigation. Residue 268 dependent steps of the complement cascade besides Clq also seems to be a good candidate. As shown in Fig. 3, its binding. A good candidate in this case is residue 274 since side chain is exposed on the outer surface of C.2 and a posi- both 3'3 and 3'4 have Gin (neutral) in this position and 3'1 tively charged His present at this residue in IgG1 and IgG3 has Lys (positive). Additional Ab-dependent steps that have is substituted by a neutral Gin in IgG4. been proposed and at which specific differences between IgG1 The present study has demonstrated that multiple amino and IgG3 have been found are the deposition of C3 and C4 acids within the C.2 domain determine the relative potency (27). of the different human IgG isotypes in complement activa- It is noteworthy that Ser331--~Pro substitution in IgG4 tion. We have shown that a single amino acid substitution or in IgG1/4 does not result in Abs as effective as IgG1 in (Ser331--~Pro) in complement-deficient IgG4 is sufficient to complement activation or Clq binding. The hierarchy of ac- impart some ability to bind Clq and activate complement

tivity is IgG1 > IgG1/4(Pro331) > IgG4(Pro331). Clearly, and that the reciprocal mutations in IgG1 and IgG3 decrease Downloaded from http://rupress.org/jem/article-pdf/178/2/661/1268014/661.pdf by guest on 30 September 2021 additional structural features of these molecules contribute their ability to bind Clq and to direct complement-mediated to their different capacities. As shown in Table 1, IgG1 and lysis. We have also produced several mutant Abs in which IgG1/4(Pro331) are identical except for C.3 and three poly- there is not a direct correlation between the ability to bind morphic residues within CH2: Tyr296Phe, Ala327Gly, and Clq and the ability to effect complement-mediated hemol- Ala330Ser. Residue 327 is mostly buried inside the molecule ysis. IgGl(Ser331), in particular, binds Clq but is incapable and is probably not directly involved in Clq binding. Residues of complement-mediated hemolysis. These mutants provide 296 and 330 are exposed on the outer surface of the CH2 do- compelling evidence for the existence of additional Ab- main (Fig. 3) and thus could be directly involved in comple- dependent steps in the classical pathway of complement acti- ment-IgG interaction. Single amino acid substitutions at res- vation. In addition, we have found that whereas it is rela- idue 296 or 330 in IgG4 however, did not have any effect tively straightforward to produce Abs with reduced or loss on complement activation or Clq binding. Also, substitu- of ability to activate complement, it much more of a chal- tion ofa Ser at position 330 in IgG1 did not decrease its ability lenge to produce genetically engineered Abs with improved to activate complement or lower its level of Clq binding. ability to activate complement. Amino acid substitutions at This result implies that the different activities of IgG1 and one location can cause distortion of local, sometimes even IgG1/4(Pro331) may be due to the combinatorial effect of distal three-dimensional structure. Thus, whereas loss of func- these polymorphic residues. Introduction of multiple mutation of mutants may support the importance of specificresidues tions at these residues should clarify this issue. as direct contacts for interacting molecules, they do not ex- The fact that IgG1/4(Pro331) is more active than IgG4- clude the possibility that the residues may function instead (Pro331) in the direct lysis assay indicates that the NH2- by influencing the conformation of the binding site located terminal part of the H chain also plays a role in determining elsewhere in the molecule. The importance of residue 331 isotype-specific differences in complement activation. The in IgG--complement interaction has been demonstrated here hinge region may contribute to this difference. In addition, by the inactivity of IgGl(Ser331) and IgG4/l(Ser331), but three residues polymorphic between IgG1 and igG4, Leu234 most convincingly by the acquisition of activity by IgG4 Phe, His268 Gin, and Lys274 Gin, are present in this portion (Pro331) and IgG1/4(Pro331). Results obtained from this type of C.2. Residue 234 is part of the lower hinge region and of functional analysis of Ab molecules will facilitate the de- has been shown to be critical for Fc receptor binding (26, sign of Igs that exhibit optimal combinations of effector func- 28). However, we found that IgG4(Leu234) is unable to trigger tions allowing for more specific and defined manipulation complement activation and the reciprocal change, IgG3 of the immune system. (Phe234), is almost as active as wild-type IgG3 (29). The

This work was supportedby U.S. PublicHealth Servicegrants CA-16858, AI-29470, and 5 T32 GM07104-18 from the National Institutes of Health, and fellowship support from Centocor. Address correspondenceto Dr. Sherie L. Morfison, Molecular Biology Institute, University of California, Los Angeles, CA 90024. Mi-Hua Tao is currently at the Institute of BiomedicalSciences, AcademiaSinica, Taipei, Taiwan 11529. Received for publication 16 April 1993.

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