HYBRIDOMA Volume 10, Number 2, 1991 Mary Ann Liebert, Inc., Publishers
Aglycosylated Chimeric Mouse/Human IgGl Antibody Retains Some Effector Function HAIMANTI DORAI,1 BARBARA M. MUELLER,2 RALPH A. REISFELD,2 and STEPHEN D. GILLIES' 'Abbott Biotech, Inc., Needham Heights. MA 02194 2Research Institute of Scripps Clinic, LaJolla, CA 92037
ABSTRACT
carbohydrate attachment site of human IgGl Ab has been eliminated by site-directed mutagenesis. Effector functions of aglycosylated Ab was then compared to its native counterpart. Aglycosylated Ab failed to exhibit any ADCC activity, but a significant level of CDC activity was retained by the aglycosylated Ab. These observations differ from those reported previously. Serum half-life and biodistribution of aglycosylated Ab in mice were comparable to the native Ab. Together, these results show that some, but not all, effector The N-linked
functions of
a
human
IgGl
Ab
are
affected
by aglycosylation.
INTRODUCTION
The most abundant isotype of human serum antibodies (Abs) is IgGl. IgGl is a glycoprotein whose N-linked carbohydrate is located in the CH2 domain of its H-chain. Many studies indicate that this carbohydrate is critical in maintaining the structure and function of IgGl (1,2). In those cases, carbohydrate were removed by enzymatic methods to study the effector functions of aglycosylated Abs. The results suggested that although effector functions were impaired, residual activity remained in most cases. The residual effector functions of aglycosylated Abs were interpreted as being due to incomplete removal of carbohydrate from the Ab molecule. Recently, Tao and Morrison (3) have used site-directed mutagenesis to remove the carbohydrate from Ig, and studies of these completely aglycosylated Abs showed that the effector functions of IgGl and IgG3 chimeric (ch) mouse/human Abs were reduced to undetectable levels. Given the ramifications of these findings on the potential use of chimeric Abs for therapy and diagnosis, we have expanded this line of inquiry to test the universality of of these results. Our extensive experience with an anti-GD2-reactive chimeric Ab (chl4.18) has facilitated the current studies in which we have eliminated the N-linked carbohydrate attachment site (Asn-297) of the above Ab by site-directed mutagenesis to elucidate the role of the carbohydrate in determining the nature and intensity of a variety of effector functions. Our results concur only in
Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; Ag, antigen; C, complement; CDC, complement dependent cytotoxicity; ch, chimeric; kbp, kilobase pairs; MTX, methotrexate.
ABBREVIATIONS:
211
part with those of behavior of
Tao and Morrison
aglycosylated
Abs will
complexes. Carbohydrate-deficient
(3) and suggest that clarification of the
require analysis
of
a
variety
of
Ab/Ag
chl4.18 Ab was properly assembled and secreted from transfected cells and bound antigen (Ag) and protein A. The biological properties of this Ab have been compared to those of its native chimeric counterpart. Aglycosylated chl4.18 Ab does not exhibit any measurable antibody dependent cellular cytotoxicity (ADCC). However, the complement dependent cytotoxicity (CDC), although it required ten times higher concentration of the antibody than the parental chl4.18 Ab for the same degree of cell lysis, was significant. Serum half-life in mice was similar for both the mutant and the native Abs, although the clearance rate for the mutant form is somewhat faster. Biodistribution of aglycosylated mutant Ig was the same as that of its native analog. These results, together with those obtained with other H-chain mutants of the chimeric anti-GD2 Ab, indicate that the carbohydrate interposed in the CH2 domain of human IgGl is necessary to maintain the appropriate structure for the retention of many but not all the effector functions of the CH2 domain. MATERIALS AND METHODS
Site-directed Mutagenesis The Hindlll-TaqI fragment (Fig. 1) containing a portion of the constant region gene of human IgGl (1.4 kbp) was cloned into M13mpl9. Site-directed mutagenesis of this fragment was performed using a synthetic oligonucleotide primer incorporating the mutation AAC—>CAG (nucleotide positions 1255-1257, Asn-297—>Gln) essentially as described (4). The mutation was confirmed by sequence analysis (5) and the mutated Hindlll-Taq I fragment was cloned into an expression vector, pdHL2 VCy, -k (12) to encode a chimeric anti-GD2 Ab (chl4.18) containing the Gln-297 mutation. Recombinant Ab Expression The Ig non-producing murine myeloma cell line Sp2/0-Agl4 was transfected with the above construct or its native counterpart by protoplast fusion (6). Media used for selection of positive clones has been described (7). Random methotrexate (MTX)-resistant clones were tested for secretion of human Ab by ELISA and cells in spent culture. One such positive clone generally secreted 1-6 was selected and the yield of Ab was improved by supplementing media with increasing concentrations of MTX up to 1 pñ. Ab was purified by chromatography
¿/g/mL/106
on
protein
A
Sepharose.
Analysis
of Effector Functions ADCC was analyzed as described (8). Two cell lines, M21 and IMR-32, that express the GD2 Ag were used as target cells. The ability of Ab to fix complement (C) and mediate complement dependent cytolysis (CDC) was measured by their ability to lyse Cr-labeled target cells in the presence of human serum as a source of C. (9). Antigen binding was analyzed by competition assays. For this purpose, a chloroform/methanol extract of membranes of IMR-32 cells was used as I as Ag (10). For serum half-life studies, recombinant Abs were labeled with described (11). Specific activity was 1 ¿/Ci per pq of Ab. The experiments were performed using groups of four animals which were sacrificed four days following I-labeled recombinant Ab. injection of
RESULTS AND DISCUSSION
Construction of
Aglycosylated
Chimeric Mouse/human Ab
The chl4.18 Ab has been used as a model system for these studies because of our extensive experience with the original chimeric Ab (12). Various derivatives of the Ab that are pertinent to the current study (13) have been generated through recombinant DNA technology. The chimeric mouse/human Ab chl4.18 is specific for GD2, a cell surface antigen of tumor cells of neuroectodermal origin (10), including the human melanoma M21 and neuroblastoma IMR-32 cell lines. The chl4.18 Ab has murine variable and human constant regions. The current studies
212
Hindu!
A.
B.
TaqI
-D
CHI
CH2
Gin 297 C C
.AGTACAACAGC 1250
FIGURE
260 Asn 297 1. Construction of the Aglycosylated Form of the Human Cyl Gene. (A) Map Hind III to Taq I fragment-contAining a portion of the genomic Cyl gene.
of the (B) The nucleotide sequence that
incorporates the
Asn mutation to Gin is shown.
involve the analysis of three mutants of the H-chain of the native chl4.18 Ab. In the first of these, Gin has been substituted for Asn-297 (Fig. 1) thereby removing the recognition sequence (Asn-X-Ser/Thr) for glycosylation (14), so that the resulting IgGl is not expected to be glycosylated. The construction of the second IgGl mutant, ACH2, in which the entire CH2 exon of IgGl has been removed has been described (13). A third mutant, CylS, in which the two hinge region cysteine residues normally involved in inter-heavy chain disulfide bond formation have been changed to serines, has also been described
(13).
Genes for each of these mutant proteins and the parental native chl4.18 Ab have been transfected into myeloma cells and secreted Ab has been purified by protein A Sepharose or anti-kappa light chain immuno-affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and reducing conditions has resolved H- and L- chains for each Ab, with the aglycosylated IgGl having a smaller apparent molecular weight than its native counterpart (Fig. 2).
:' *
VJ < 67 Kd
ii&m
43 Kd
M
B
Polyacrylamide Gel Electrophoresis of Aglycosylated Ab and its Native Ten micrograms of each Ab was subjected to a 7.5% SDS-PAGE. Lane 1, aglycosylated Ab; lane 2, native Ab treated with 0.3 U of N-Glycanase (Genzyme) for 18 h at 37°C; lane c, native Ab. Only the H-chain is shown. FIGURE 2.
Counterpart.
213
Antigen Binding IgGl
and Effector Functions of
Aglycosylated
The antigen binding activities of aglycosylated and native chimeric Abs were tested in a competitive binding assay. As seen in Figure 3, both of the unlabeled Abs competed to the same extent with the peroxidase-labeled native chl4.18 Ab for the Ag showing that binding is unchanged for the mutant IgGl Ab. 80-, -
7060-•
8
S0"
¡40-
rA Vk-
X t3
M 30--
2010-
0-I-v 10 100 ng/WELL
1000 UNLABELED ANTIBODY
1
ng/WELL
10 100 UNLABELED ANTIBODY
FIGURE 3. Competitive Antigen Binding Analysis of ch 14.18 and Aglycoslated Mutant Antibodies. (A) Two hr incubation of tracer and test Abs at 37°C. (O), ch 14.18 Ab; (O), aglycosylated Ab (B) Eighteen hr. incubation of Abs at 4°C. (0), chl4.18 Ab; ( ), aglycosylated Ab. The amount of bound tracer (a horseradish peroxidase-conjugated chl4.18 Ab) was determined in the absence of competitor to give 100% binding value.
Previous studies have shown that the antigen binding activity of the ACH2 significantly increased although the overall affinity is not (13). This suggested that interactions between the CH2 exon and the Fab (which have been reported in crystallographic studies, ref.14), influence the rate of Ab/Ag binding. Removal of the carbohydrate changes the three-dimensional structure of the CH2 exon (15) so that alteration of the rate of binding of the aglycosylated antibody to the antigen might also be expected. No change in the rate of binding of the aglycosylated Ab to the antigen has been observed. Apparently, removal of just the carbohydrate from the CH2 exon does not alter the three-dimensional structure enough to perturb the interaction between the Fab and CH2 domains. The ability of aglycosylated Ab to mediate complement-dependent lysis of melanoma target cells was found to be ten-fold less than that of the parental chl4.18 Ab (Fig. 4A), i.e., the same degree of cell lysis could be obtained but at a ten-fold higher antibody concentration. In this respect, the Gln-297 mutant Ab is similar to the CylS mutant Ab (13). Our results are in agreement with studies of Nose and Wigzell (16) who have shown with an assay similar to ours that although carbohydrate-depleted mouse monoclonal IgG has a decreased ability to activate complement, high concentrations of these Abs did activate complement and mediate C-dependent lysis. Binding of Clq is localized in the CH2 domain, and it is possible that the carbohydrate moity changes the conformation of the CH2 domain such that aglycosylated Ab has a reduced ability to bind Clq. This reduced binding would be reflected in the ten-fold reduction in CDC activity of the aglycosylated Ab. In this regard, our results differ from those of Tao and Morrison (3) who have found that aglycosylated IgGl completely loses its ability to bind Clq. This discrepancy can possibly be attributed to the nature of binding by the Ab which differs for each Ab/Ag complex and to the increased sensitivity of our assay which measures the complement mediated lysis of target cells. In contrast to the CDC assay, our results on ADCC are in agreement with those of Tao and Morrison (3). Figure 4 shows the results of a typical ADCC assay of mutant of chl4.18 is
214
100 ANTIBODY
1000
1000
10 100 ANTIBODY (ng/mL)
(rrç/mL)
Effector Functions of Aglycosylated Ab and its Native Counterpart. (A) Complement-dependent cytotoxicity (CDC) activity of chl4.18, aglycosylated
FIGURE 4.
chl4.18/IgG4 Abs. The specific lysis of M21 cells in the presence of human complement and the indicated amounts of Abs are shown for chl4.18 (0), Aglycosylated (A) Abs and chl4.18/IgG4 (fl ). (B) Antibody dependent cellular cytotoxicity (ADCC) of human peripheral blood mononuclear cells mediated by aglycosylated Ab and its native counterpart. The specific lysis of M21 human and
melanoma cells in the presence of the indicated concentrations of chl4.18 (0), aglycosylated (A) and chl4.18/IgG4 (n ) Abs are shown for an effector target ratio of 100:1. The average of triplicate samples is shown for each data point.
50 •
O O m
ch14.18
aglyco ch14.18
40 a
ch14.18-ACH2
o
v>
o
O
O) o O)
40 Time
60
100
80
(hours)
FIGURE 5. Blood Clearance of Ab Following i.v. Injection into Bearing M21 Tumors. Each animal was injected with 25 pq
Mice
1251-labelled
Athymic (nu/nu) (3 ¿/Ci )
or
Ab and bled 0.5, 2, 4, 8, 24, 72, and 96 hrs thereafter. Data is given as percent injected dose per milliliter of blood. Each point and error bar represents the mean and standard deviation for four animals; chl4.18 (8), aglycosylated (a) and chl4.18-ACH2 (A).
215
aglycosylated IgGl in which its ability to mediate the lysis of Ag-positive tumor cells is compared to that of its native counterpart. The loss of ADCC activity upon aglycosylation of the IgGl of the chl4.18 Ab likely reflects its failure to bind the Fc receptor. In our previous studies (13), we found that the removal of the CH2 domain led to a loss of ADCC activity, confirming that the binding of the high affinity receptor can be mapped to the CH2 domain. Duncan et al. have used site-directed mutagenesis to show that the binding site for the high affinity Fc
receptor is localized in the CH2 exon (17). On the other hand, the CylS derivative of the chl4.18 Ab also has very little ADCC activity although it contains an intact glycosylated CH2 exon (13). This observation lends support to the notion that maintenance of the structure of the Ab is necessary for the CH2 domain to bind the Fc receptor. Consequently, mutations that perturb this structure, i.e., changes in the carbohydrate interposed between the paired CH2 domains or the unpinning of the hinge, lead to an inability to bind Fc receptor and hence to the loss of ADCC activity. Blood clearance studies in mice show similar kinetics for the Gln-297 Ab as for its native counterpart (Fig. 5), although the rate at which the aglycosylated Ab is cleared is somewhat faster. In comparison, the ACH2 mutant is cleared much more rapidly from the blood. The half-lives of the aglycosylated and the native Abs are approximately five days whereas that of ACH2 is 12 hours. Biodistribution studies also indicate no significant differences between the aglycosylated and native chl4.18 Abs (data not shown). However, serum clearance studies of these chimeric Abs in humans may yield results that differ from those obtained in mice. Taken together, our results indicate that carbohydrates do not play an important role in the catabolism of human Ab in mice. Instead, Ab clearance is determined by the presence or absence of the CH2 domain as reported earlier (19).
ACKNOWLEDGMENTS We thank Dr. K.-M. Lo for synthetic oligonucleotides, J. Wesolowski for ADCC and CDC assay and Dr. R.M. Reilly for critical reading of the manuscript.
REFERENCES
MARGHERITA,S., TOKUDA S. and MESSNER, R.P. (1973). Studies of biologic and sérologie activities of rabbit-IgG antibody depleted of carbohydrate residues. J. Immunol. Ill, 1690-1698.
1.
WILLIAMS, R., OSTERLAND, C.K.,
2.
WINKELHAKE, J.L., and NICHOLSON. G.L (1976). Chem. 251, 1074-1081.
3.
TAO, M.H., and Morrison, S.L. (1989). Studies of mouse-human IgG. J. Immunol. 143, 2595-2601.
4.
ZOLLER, M.J., and SMITH, DNA
5.
fragments
aglycosylated
chimeric
(1983). Oligonucleotide-directed mutagenesis of Enzymol. 100, 468-472.
cloned into M13 vectors. Methods
(1977).
SANGER, F., NICKLIN, S., and COULSON,
A.R.
DNA
inhibitors. Proc. Nati. Acad. Sei. USA
sequencing
74, 5463-5467. 6.
M.
J. Biol.
Aglycosylantibody.
with
chain-terminating
GILLIES, S.D., MORRISON, S.L., 01, V.T., and TONEGAWA,
S.
(1983).
A
tissue-specific transcriptional enhancer element is located in the major intron of a rearranged immunoglobulin heavy-chain gene. Cell 33, 717-728
7.
GILLIES, S.D., DORAI, H., WESOLOWSKI, J.S., MAJEAU, G., YOUNG, D., BOYD, J., GARDNER, J., and JAMES, K. (1989). Expression of anti-tetanus toxoid antibody in transfected murine myeloma cells. Biotechnology 7, 799-804.
8.
WESOLOWSKI, J.S., Ault,K., HOUGHTON, R., inhibition of
antibody-dependent
cell
216
and PARKMAN, R. (1984). Heparin Exp. Hematol. 12,700-705.
lysis.
9.
GRABSTEIN, K., and CHEN, Y.U. (1980). Cell-mediated cystolytic responses. In: B.B. Mishel and S.M. Shiigi (Eds.), "Selected Methods in Cellular Immunology". W.H. Freeman and Co., San Francisco, CA., pp. 128-135.
10.
MUJOO, K., CHERESH, D.A., YANG, H.A., and REISFELD,
(1987).
R.A.
Disialoganglioside GD2 on neuroblastoma cells: target antigen for monoclonal Ab-mediated cytolysis and suppression of tumor growth. Cancer Res. 47, 1098-1104.
11.
MUELLER, B.M., ROMERDAHL, C.A, GILLIES, S.D., REISFELD, Enhancement of antibody-dependent cytoxicity with antibody. J. Immunol. 144, 1382-1386.
12.
a
R.A.
(1990).
chimeric anti-GD2
J.S. (1989). High level of chimeric antibodies using adapted cDNA variable region cassettes. J. Immunol. Meth. 125, 191-202.
GILLIES, S.D., Lo, K.-M., and WESOLOWSKI,
expression 13.
GILLIES, S.D., and WESOLOWSKI, J.S. (1990). Antigen binding and biological activities of Hum. Antibod.
14.
engineered mutant chimeric Hybridomas. 1, 47-54.
specificities.
AMZEL, L.M. and POLJAK, R.J. (1979). Three dimensinal structure of
immunoglobulins.
15.
Abs with human tumor
Ann. Rev. Biochem.
48, 961-967.
RADEMACHER, T.W., and DEWK, R.A. (1984). Structural, functional and
analysis of immunoglobulin G-derived asparagine-linked oligosaccharides. Prog. Immunol. 5, 95-112. conformational
16.
NOSE, M., and WIGZELL, chains
17.
on
H.
(1983). Biological significance of carbohydrate Proc. Nati. Acad. Sei. USA 80, 6632-6636.
monoclonal antibodies.
DUNCAN, A.R., WOOF, J.M., PARTRIDGE,
L.J.
BURTON, D.R. and WINTER, G.
(1988). Localization of the binding site for the high-affinity on IgG. Nature. 332, 563-565.
18.
WINZLER,
R.J.
receptor
(1973). The chemistry of glycoproteins.
"Hormonal Proteins and 19.
Fc
Peptides,"
In: H. Li (Ed.), Vol. I. Academic Press, N.Y., pp. 1-15.
MUELLER, B.M., REISFELD, R.A., and GILLIES, S.D. (1990). Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2. Proc. Nati. Acad. Sei. USA. in press. Address
reprint requests
to
Stephen D. Gillies Abbott Biotech, Inc. 119 Fourth Avenue Needham Heights, MA 02194 Dr.
Received for publication September 10, 1990 November 30, 1990
Accepted
217
Aglycosylated Chimeric Mouse/Human IgGl Antibody Retains Some Effector Function HAIMANTI DORAI,1 BARBARA M. MUELLER,2 RALPH A. REISFELD,2 and STEPHEN D. GILLIES' 'Abbott Biotech, Inc., Needham Heights. MA 02194 2Research Institute of Scripps Clinic, LaJolla, CA 92037
ABSTRACT
carbohydrate attachment site of human IgGl Ab has been eliminated by site-directed mutagenesis. Effector functions of aglycosylated Ab was then compared to its native counterpart. Aglycosylated Ab failed to exhibit any ADCC activity, but a significant level of CDC activity was retained by the aglycosylated Ab. These observations differ from those reported previously. Serum half-life and biodistribution of aglycosylated Ab in mice were comparable to the native Ab. Together, these results show that some, but not all, effector The N-linked
functions of
a
human
IgGl
Ab
are
affected
by aglycosylation.
INTRODUCTION
The most abundant isotype of human serum antibodies (Abs) is IgGl. IgGl is a glycoprotein whose N-linked carbohydrate is located in the CH2 domain of its H-chain. Many studies indicate that this carbohydrate is critical in maintaining the structure and function of IgGl (1,2). In those cases, carbohydrate were removed by enzymatic methods to study the effector functions of aglycosylated Abs. The results suggested that although effector functions were impaired, residual activity remained in most cases. The residual effector functions of aglycosylated Abs were interpreted as being due to incomplete removal of carbohydrate from the Ab molecule. Recently, Tao and Morrison (3) have used site-directed mutagenesis to remove the carbohydrate from Ig, and studies of these completely aglycosylated Abs showed that the effector functions of IgGl and IgG3 chimeric (ch) mouse/human Abs were reduced to undetectable levels. Given the ramifications of these findings on the potential use of chimeric Abs for therapy and diagnosis, we have expanded this line of inquiry to test the universality of of these results. Our extensive experience with an anti-GD2-reactive chimeric Ab (chl4.18) has facilitated the current studies in which we have eliminated the N-linked carbohydrate attachment site (Asn-297) of the above Ab by site-directed mutagenesis to elucidate the role of the carbohydrate in determining the nature and intensity of a variety of effector functions. Our results concur only in
Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; Ag, antigen; C, complement; CDC, complement dependent cytotoxicity; ch, chimeric; kbp, kilobase pairs; MTX, methotrexate.
ABBREVIATIONS:
211
part with those of behavior of
Tao and Morrison
aglycosylated
Abs will
complexes. Carbohydrate-deficient
(3) and suggest that clarification of the
require analysis
of
a
variety
of
Ab/Ag
chl4.18 Ab was properly assembled and secreted from transfected cells and bound antigen (Ag) and protein A. The biological properties of this Ab have been compared to those of its native chimeric counterpart. Aglycosylated chl4.18 Ab does not exhibit any measurable antibody dependent cellular cytotoxicity (ADCC). However, the complement dependent cytotoxicity (CDC), although it required ten times higher concentration of the antibody than the parental chl4.18 Ab for the same degree of cell lysis, was significant. Serum half-life in mice was similar for both the mutant and the native Abs, although the clearance rate for the mutant form is somewhat faster. Biodistribution of aglycosylated mutant Ig was the same as that of its native analog. These results, together with those obtained with other H-chain mutants of the chimeric anti-GD2 Ab, indicate that the carbohydrate interposed in the CH2 domain of human IgGl is necessary to maintain the appropriate structure for the retention of many but not all the effector functions of the CH2 domain. MATERIALS AND METHODS
Site-directed Mutagenesis The Hindlll-TaqI fragment (Fig. 1) containing a portion of the constant region gene of human IgGl (1.4 kbp) was cloned into M13mpl9. Site-directed mutagenesis of this fragment was performed using a synthetic oligonucleotide primer incorporating the mutation AAC—>CAG (nucleotide positions 1255-1257, Asn-297—>Gln) essentially as described (4). The mutation was confirmed by sequence analysis (5) and the mutated Hindlll-Taq I fragment was cloned into an expression vector, pdHL2 VCy, -k (12) to encode a chimeric anti-GD2 Ab (chl4.18) containing the Gln-297 mutation. Recombinant Ab Expression The Ig non-producing murine myeloma cell line Sp2/0-Agl4 was transfected with the above construct or its native counterpart by protoplast fusion (6). Media used for selection of positive clones has been described (7). Random methotrexate (MTX)-resistant clones were tested for secretion of human Ab by ELISA and cells in spent culture. One such positive clone generally secreted 1-6 was selected and the yield of Ab was improved by supplementing media with increasing concentrations of MTX up to 1 pñ. Ab was purified by chromatography
¿/g/mL/106
on
protein
A
Sepharose.
Analysis
of Effector Functions ADCC was analyzed as described (8). Two cell lines, M21 and IMR-32, that express the GD2 Ag were used as target cells. The ability of Ab to fix complement (C) and mediate complement dependent cytolysis (CDC) was measured by their ability to lyse Cr-labeled target cells in the presence of human serum as a source of C. (9). Antigen binding was analyzed by competition assays. For this purpose, a chloroform/methanol extract of membranes of IMR-32 cells was used as I as Ag (10). For serum half-life studies, recombinant Abs were labeled with described (11). Specific activity was 1 ¿/Ci per pq of Ab. The experiments were performed using groups of four animals which were sacrificed four days following I-labeled recombinant Ab. injection of
RESULTS AND DISCUSSION
Construction of
Aglycosylated
Chimeric Mouse/human Ab
The chl4.18 Ab has been used as a model system for these studies because of our extensive experience with the original chimeric Ab (12). Various derivatives of the Ab that are pertinent to the current study (13) have been generated through recombinant DNA technology. The chimeric mouse/human Ab chl4.18 is specific for GD2, a cell surface antigen of tumor cells of neuroectodermal origin (10), including the human melanoma M21 and neuroblastoma IMR-32 cell lines. The chl4.18 Ab has murine variable and human constant regions. The current studies
212
Hindu!
A.
B.
TaqI
-D
CHI
CH2
Gin 297 C C
.AGTACAACAGC 1250
FIGURE
260 Asn 297 1. Construction of the Aglycosylated Form of the Human Cyl Gene. (A) Map Hind III to Taq I fragment-contAining a portion of the genomic Cyl gene.
of the (B) The nucleotide sequence that
incorporates the
Asn mutation to Gin is shown.
involve the analysis of three mutants of the H-chain of the native chl4.18 Ab. In the first of these, Gin has been substituted for Asn-297 (Fig. 1) thereby removing the recognition sequence (Asn-X-Ser/Thr) for glycosylation (14), so that the resulting IgGl is not expected to be glycosylated. The construction of the second IgGl mutant, ACH2, in which the entire CH2 exon of IgGl has been removed has been described (13). A third mutant, CylS, in which the two hinge region cysteine residues normally involved in inter-heavy chain disulfide bond formation have been changed to serines, has also been described
(13).
Genes for each of these mutant proteins and the parental native chl4.18 Ab have been transfected into myeloma cells and secreted Ab has been purified by protein A Sepharose or anti-kappa light chain immuno-affinity chromatography. Polyacrylamide gel electrophoresis under denaturing and reducing conditions has resolved H- and L- chains for each Ab, with the aglycosylated IgGl having a smaller apparent molecular weight than its native counterpart (Fig. 2).
:' *
VJ < 67 Kd
ii&m
43 Kd
M
B
Polyacrylamide Gel Electrophoresis of Aglycosylated Ab and its Native Ten micrograms of each Ab was subjected to a 7.5% SDS-PAGE. Lane 1, aglycosylated Ab; lane 2, native Ab treated with 0.3 U of N-Glycanase (Genzyme) for 18 h at 37°C; lane c, native Ab. Only the H-chain is shown. FIGURE 2.
Counterpart.
213
Antigen Binding IgGl
and Effector Functions of
Aglycosylated
The antigen binding activities of aglycosylated and native chimeric Abs were tested in a competitive binding assay. As seen in Figure 3, both of the unlabeled Abs competed to the same extent with the peroxidase-labeled native chl4.18 Ab for the Ag showing that binding is unchanged for the mutant IgGl Ab. 80-, -
7060-•
8
S0"
¡40-
rA Vk-
X t3
M 30--
2010-
0-I-v 10 100 ng/WELL
1000 UNLABELED ANTIBODY
1
ng/WELL
10 100 UNLABELED ANTIBODY
FIGURE 3. Competitive Antigen Binding Analysis of ch 14.18 and Aglycoslated Mutant Antibodies. (A) Two hr incubation of tracer and test Abs at 37°C. (O), ch 14.18 Ab; (O), aglycosylated Ab (B) Eighteen hr. incubation of Abs at 4°C. (0), chl4.18 Ab; ( ), aglycosylated Ab. The amount of bound tracer (a horseradish peroxidase-conjugated chl4.18 Ab) was determined in the absence of competitor to give 100% binding value.
Previous studies have shown that the antigen binding activity of the ACH2 significantly increased although the overall affinity is not (13). This suggested that interactions between the CH2 exon and the Fab (which have been reported in crystallographic studies, ref.14), influence the rate of Ab/Ag binding. Removal of the carbohydrate changes the three-dimensional structure of the CH2 exon (15) so that alteration of the rate of binding of the aglycosylated antibody to the antigen might also be expected. No change in the rate of binding of the aglycosylated Ab to the antigen has been observed. Apparently, removal of just the carbohydrate from the CH2 exon does not alter the three-dimensional structure enough to perturb the interaction between the Fab and CH2 domains. The ability of aglycosylated Ab to mediate complement-dependent lysis of melanoma target cells was found to be ten-fold less than that of the parental chl4.18 Ab (Fig. 4A), i.e., the same degree of cell lysis could be obtained but at a ten-fold higher antibody concentration. In this respect, the Gln-297 mutant Ab is similar to the CylS mutant Ab (13). Our results are in agreement with studies of Nose and Wigzell (16) who have shown with an assay similar to ours that although carbohydrate-depleted mouse monoclonal IgG has a decreased ability to activate complement, high concentrations of these Abs did activate complement and mediate C-dependent lysis. Binding of Clq is localized in the CH2 domain, and it is possible that the carbohydrate moity changes the conformation of the CH2 domain such that aglycosylated Ab has a reduced ability to bind Clq. This reduced binding would be reflected in the ten-fold reduction in CDC activity of the aglycosylated Ab. In this regard, our results differ from those of Tao and Morrison (3) who have found that aglycosylated IgGl completely loses its ability to bind Clq. This discrepancy can possibly be attributed to the nature of binding by the Ab which differs for each Ab/Ag complex and to the increased sensitivity of our assay which measures the complement mediated lysis of target cells. In contrast to the CDC assay, our results on ADCC are in agreement with those of Tao and Morrison (3). Figure 4 shows the results of a typical ADCC assay of mutant of chl4.18 is
214
100 ANTIBODY
1000
1000
10 100 ANTIBODY (ng/mL)
(rrç/mL)
Effector Functions of Aglycosylated Ab and its Native Counterpart. (A) Complement-dependent cytotoxicity (CDC) activity of chl4.18, aglycosylated
FIGURE 4.
chl4.18/IgG4 Abs. The specific lysis of M21 cells in the presence of human complement and the indicated amounts of Abs are shown for chl4.18 (0), Aglycosylated (A) Abs and chl4.18/IgG4 (fl ). (B) Antibody dependent cellular cytotoxicity (ADCC) of human peripheral blood mononuclear cells mediated by aglycosylated Ab and its native counterpart. The specific lysis of M21 human and
melanoma cells in the presence of the indicated concentrations of chl4.18 (0), aglycosylated (A) and chl4.18/IgG4 (n ) Abs are shown for an effector target ratio of 100:1. The average of triplicate samples is shown for each data point.
50 •
O O m
ch14.18
aglyco ch14.18
40 a
ch14.18-ACH2
o
v>
o
O
O) o O)
40 Time
60
100
80
(hours)
FIGURE 5. Blood Clearance of Ab Following i.v. Injection into Bearing M21 Tumors. Each animal was injected with 25 pq
Mice
1251-labelled
Athymic (nu/nu) (3 ¿/Ci )
or
Ab and bled 0.5, 2, 4, 8, 24, 72, and 96 hrs thereafter. Data is given as percent injected dose per milliliter of blood. Each point and error bar represents the mean and standard deviation for four animals; chl4.18 (8), aglycosylated (a) and chl4.18-ACH2 (A).
215
aglycosylated IgGl in which its ability to mediate the lysis of Ag-positive tumor cells is compared to that of its native counterpart. The loss of ADCC activity upon aglycosylation of the IgGl of the chl4.18 Ab likely reflects its failure to bind the Fc receptor. In our previous studies (13), we found that the removal of the CH2 domain led to a loss of ADCC activity, confirming that the binding of the high affinity receptor can be mapped to the CH2 domain. Duncan et al. have used site-directed mutagenesis to show that the binding site for the high affinity Fc
receptor is localized in the CH2 exon (17). On the other hand, the CylS derivative of the chl4.18 Ab also has very little ADCC activity although it contains an intact glycosylated CH2 exon (13). This observation lends support to the notion that maintenance of the structure of the Ab is necessary for the CH2 domain to bind the Fc receptor. Consequently, mutations that perturb this structure, i.e., changes in the carbohydrate interposed between the paired CH2 domains or the unpinning of the hinge, lead to an inability to bind Fc receptor and hence to the loss of ADCC activity. Blood clearance studies in mice show similar kinetics for the Gln-297 Ab as for its native counterpart (Fig. 5), although the rate at which the aglycosylated Ab is cleared is somewhat faster. In comparison, the ACH2 mutant is cleared much more rapidly from the blood. The half-lives of the aglycosylated and the native Abs are approximately five days whereas that of ACH2 is 12 hours. Biodistribution studies also indicate no significant differences between the aglycosylated and native chl4.18 Abs (data not shown). However, serum clearance studies of these chimeric Abs in humans may yield results that differ from those obtained in mice. Taken together, our results indicate that carbohydrates do not play an important role in the catabolism of human Ab in mice. Instead, Ab clearance is determined by the presence or absence of the CH2 domain as reported earlier (19).
ACKNOWLEDGMENTS We thank Dr. K.-M. Lo for synthetic oligonucleotides, J. Wesolowski for ADCC and CDC assay and Dr. R.M. Reilly for critical reading of the manuscript.
REFERENCES
MARGHERITA,S., TOKUDA S. and MESSNER, R.P. (1973). Studies of biologic and sérologie activities of rabbit-IgG antibody depleted of carbohydrate residues. J. Immunol. Ill, 1690-1698.
1.
WILLIAMS, R., OSTERLAND, C.K.,
2.
WINKELHAKE, J.L., and NICHOLSON. G.L (1976). Chem. 251, 1074-1081.
3.
TAO, M.H., and Morrison, S.L. (1989). Studies of mouse-human IgG. J. Immunol. 143, 2595-2601.
4.
ZOLLER, M.J., and SMITH, DNA
5.
fragments
aglycosylated
chimeric
(1983). Oligonucleotide-directed mutagenesis of Enzymol. 100, 468-472.
cloned into M13 vectors. Methods
(1977).
SANGER, F., NICKLIN, S., and COULSON,
A.R.
DNA
inhibitors. Proc. Nati. Acad. Sei. USA
sequencing
74, 5463-5467. 6.
M.
J. Biol.
Aglycosylantibody.
with
chain-terminating
GILLIES, S.D., MORRISON, S.L., 01, V.T., and TONEGAWA,
S.
(1983).
A
tissue-specific transcriptional enhancer element is located in the major intron of a rearranged immunoglobulin heavy-chain gene. Cell 33, 717-728
7.
GILLIES, S.D., DORAI, H., WESOLOWSKI, J.S., MAJEAU, G., YOUNG, D., BOYD, J., GARDNER, J., and JAMES, K. (1989). Expression of anti-tetanus toxoid antibody in transfected murine myeloma cells. Biotechnology 7, 799-804.
8.
WESOLOWSKI, J.S., Ault,K., HOUGHTON, R., inhibition of
antibody-dependent
cell
216
and PARKMAN, R. (1984). Heparin Exp. Hematol. 12,700-705.
lysis.
9.
GRABSTEIN, K., and CHEN, Y.U. (1980). Cell-mediated cystolytic responses. In: B.B. Mishel and S.M. Shiigi (Eds.), "Selected Methods in Cellular Immunology". W.H. Freeman and Co., San Francisco, CA., pp. 128-135.
10.
MUJOO, K., CHERESH, D.A., YANG, H.A., and REISFELD,
(1987).
R.A.
Disialoganglioside GD2 on neuroblastoma cells: target antigen for monoclonal Ab-mediated cytolysis and suppression of tumor growth. Cancer Res. 47, 1098-1104.
11.
MUELLER, B.M., ROMERDAHL, C.A, GILLIES, S.D., REISFELD, Enhancement of antibody-dependent cytoxicity with antibody. J. Immunol. 144, 1382-1386.
12.
a
R.A.
(1990).
chimeric anti-GD2
J.S. (1989). High level of chimeric antibodies using adapted cDNA variable region cassettes. J. Immunol. Meth. 125, 191-202.
GILLIES, S.D., Lo, K.-M., and WESOLOWSKI,
expression 13.
GILLIES, S.D., and WESOLOWSKI, J.S. (1990). Antigen binding and biological activities of Hum. Antibod.
14.
engineered mutant chimeric Hybridomas. 1, 47-54.
specificities.
AMZEL, L.M. and POLJAK, R.J. (1979). Three dimensinal structure of
immunoglobulins.
15.
Abs with human tumor
Ann. Rev. Biochem.
48, 961-967.
RADEMACHER, T.W., and DEWK, R.A. (1984). Structural, functional and
analysis of immunoglobulin G-derived asparagine-linked oligosaccharides. Prog. Immunol. 5, 95-112. conformational
16.
NOSE, M., and WIGZELL, chains
17.
on
H.
(1983). Biological significance of carbohydrate Proc. Nati. Acad. Sei. USA 80, 6632-6636.
monoclonal antibodies.
DUNCAN, A.R., WOOF, J.M., PARTRIDGE,
L.J.
BURTON, D.R. and WINTER, G.
(1988). Localization of the binding site for the high-affinity on IgG. Nature. 332, 563-565.
18.
WINZLER,
R.J.
receptor
(1973). The chemistry of glycoproteins.
"Hormonal Proteins and 19.
Fc
Peptides,"
In: H. Li (Ed.), Vol. I. Academic Press, N.Y., pp. 1-15.
MUELLER, B.M., REISFELD, R.A., and GILLIES, S.D. (1990). Serum half-life and tumor localization of a chimeric antibody deleted of the CH2 domain and directed against the disialoganglioside GD2. Proc. Nati. Acad. Sei. USA. in press. Address
reprint requests
to
Stephen D. Gillies Abbott Biotech, Inc. 119 Fourth Avenue Needham Heights, MA 02194 Dr.
Received for publication September 10, 1990 November 30, 1990
Accepted
217
