0013-7227/90/1266-2773$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
Identification of the Insulin-Like Growth Factor I (IGF I) Epitopes Recognized by Monoclonal and Polyclonal Antibodies to IGF I MARGARET A. CASCIERI,* MARVIN L. BAYNE, ELZBIETA BER, BARBARA G. GREEN, GINA W. MEN, AND GARY G. CHICCHI Departments of Biochemical Endocrinology and Growth Factor Research (M.L.B.), Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey, 07065
binding of 125I-IGF I to type 1 receptors on human placental membranes. In addition, SM 1.2 inhibits the ability of IGF I and IGF analogs for which it has high affinity to stimulate DNA synthesis in murine fibroblasts. In contrast, analogs with substitutions at residues 15 and 16, which have poor affinity for SM 1.2, stimulate DNA synthesis with equal potency in the presence and absence of SM 1.2. These antibodies bind normally to analogs which we have previously shown have drastically reduced binding to type 1 IGF receptors, indicating that the antibodies and the receptors recognize distinct domains of IGF I. {Endocrinology 126: 2773-2777, 1990)
ABSTRACT. We have characterized the binding epitopes of human insulin-like growth factor I (IGF I) for a polyclonal (UB286) and a monoclonal (SM 1.2) antibody using IGF analogs obtained by site-directed mutagenesis. The polyclonal antibody, UB286, which was obtained from the National Hormone and Pituitary Program, recognizes determinants surrounding residues 15 and 16 in the B-region and residues 49-51, 55 and 56 in the A-region. These residues are predicted to be within helical segments which are accessible for surface binding. The monoclonal antibody SM 1.2 selectively recognizes the region surrounding residues 15 and 16. Antibodies UB286 and SM 1.2 are both neutralizing antibodies as judged by their ability to inhibit
I
NSULIN-LIKE growth factor I (IGF I) is a polypep-
a high affinity polyclonal antibody to IGF I in 1977.
tide growth factor that has important hormonal and autocrine effects on various tissues. It is a member of a family of peptides that includes insulin and IGF II (1, 2). The amino terminal 29 amino acids of IGF I are homologous to the B-chain of insulin. The next 12 amino acids (C-region) link the B-region with the A-region (residues 42-62), which is homologous to the A-chain of insulin. The carboxyl terminus of IGF I is extended by eight amino acids to form the D-region. The biological effects of IGF I are mediated through the type 1 IGF receptor (3), although IGF I also binds to insulin receptors (4) and type 2 IGF receptors (5) with lower affinity. In addition, IGF I, but not insulin, interacts with high affinity with a family of soluble binding proteins (IGF-BP), several of which have recently been cloned (6-9). These specific binding proteins are found in serum and in the conditioned media of various cell types, and they modify IGF I activity in a complex manner (10-14). Furlanetto et al. (15) first described the preparation of
Since then, a series of polyclonal antibodies to IGF I prepared by Van Wyk and his colleagues have been widely used throughout the endocrine research community via distribution by the Pituitary Hormone Program at NIDDK. This same group has recently prepared a neutralizing monoclonal antibody to IGF I (16). At the present time, it is not known which epitopes of IGF I are involved in the binding of these antibodies to IGF I. We have used site-directed mutagenesis to determine the domains of IGF I which are involved in binding to the receptors and soluble binding proteins (17-20). The tyrosine residue at position 24 and residues between 28 and 37 in the C-region are involved in binding to the type 1 IGF receptor (17, 18). Substitutions at the amino terminus and within the first helical region of IGF I with the analogous residues in insulin selectively alter binding to the soluble binding proteins in serum (19) and in the conditioned media of various cell lines (12). Substitutions of the surface residues in the two helices in the A-region between residues 42 and 60 with the analogous residues in insulin alter binding of IGF I to the type 2 IGF receptor (20) and to IGF-BP-1 (21). In the present series of experiments, we have used these IGF analogs to map the binding epitopes for a polyclonal and a monoclonal antibody to IGF I.
Received December 11, 1989. Address all correspondence and requests for reprints to: Dr. M.A. Cascieri, Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065.
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IDENTIFICATION OF IGF I ANTIBODY BINDING EPITOPES
2774
Materials and Methods Preparation of IGF analogs Analogs were prepared as previously described (17-20). Briefly, site-directed mutagenesis was performed using a synthetic gene encoding IGF I; the genes were expressed in Saccharomyces cerevisiae using a vector encoding the yeast pre-pro al protein sequence, and the secreted proteins were purified in three steps. The purity of the proteins was confirmed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining, and the mass was determined using amino acid analysis. RIAs Antibody UB286. 125I-IGF I was prepared as previously described (22). UB286 (1:8000 final dilution) and competing peptides were incubated in 0.4 ml 0.03 M sodium phosphate, pH 7.5 containing 0.01 M EDTA, 0.02% protamine sulfate, 0.05% Tween 20, and 0.02% sodium azide. After 1 h at 22 C, 125I-IGF I (1.5 X 104 cpm) was added in 0.1 ml normal rabbit serum (1:750 final dilution). After 20 h at 4 C, 0.05 ml sheep antirabbit 7-globulin (Organon Teknika Cappel, West Chester, PA, 1:25 dilution) and 0.1 ml 20% polyethylene glycol were added. After 1 h at 22 C, samples were diluted with 1 ml assay buffer and centrifuged for 10 min at 4 C at 4000 rpm (Beckman J6B centrifuge, Beckman Instruments, Fullerton, CA). The supernatant was removed and the pellets were counted. In control experiments, normal rabbit serum (1:750) was incubated with 125 I-IGF I in the presence or absence of antibody as described above. Charcoal was added to separate free from bound ligand as described previously (17). Under these conditions, charcoal precipitation removed 70% or more than 99% of the ligand in the presence or absence of antisera, respectively. Thus, under these conditions, virtually none of the ligand bound to the small amounts of IGF-BP present in the serum.
Endo • 1990 Vol 126 • No 6
ml 0.1 N NaOH with 2% Na2CO3 was added, and cell digests were counted. Results We determined the ability of 12 analogs of IGF I to inhibit the binding of 125I-IGF I to the polyclonal antibody UB286 and to the monoclonal antibody SM 1.2. IGF I inhibits the binding to UB286 and SM 1.2 with an IC50 = 0.030 nM and 2.5 nM, respectively. The analog in which the first 16 amino acids of IGF I were replaced with the first 17 amino acids of the B-chain of insulin (B-chain mutant) has more than 100-fold lower affinity for both antibodies (Fig. 1). [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I and [Tyr 15, Leu 16] IGF I, two analogs in which residues 15 and 16 of IGF I are replaced by the analogous residues in insulin, have 25-fold and 100-fold lower affinity for UB286 and 100-fold and 25-fold lower affinity for SM 1.2, respectively (Fig. 1). In contrast, [Gin 3, Ala 4] IGF I, the analog in which only the amino
100 -
Antibody SM 1.2. Antibody was purified from ascites (Developmental Hybridoma Bank, Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, MD) using a 1-ml Protein G-sepharose 4 fast flow column (Pharmacia/LKB, Piscataway, NJ) as per manufacturer's instructions. Antibody (100 pM final concentration), 125IIGF I (1 X 104 cpm), and competing peptides were incubated in 0.5 ml 0.2 M sodium phosphate, pH 7.5, containing 0.25% BSA and 0.1% bovine 7-globulin (Pentex fraction II, ICN Immuno Biologicals, Lisle, IL). After 20 h at 4 C, added 0.5 ml 25% polyethylene glycol and centrifuged as above. DNA synthesis assays. Assays were performed in L7 murine fibroblasts as previously described (22) except for the following changes. Cells (2 x 105/\vell) were plated onto 96-well plates. After 6 h and 24 h, media were replaced with Dulbecco's modified Eagle's medium containing 0.1% calf serum. After 44 h, the media were replaced as above, and peptides and antibody were added. After 52 h, 3H thymidine (0.4 /^Ci, 25 nM) was added. After 70 h, the media were removed, cells were washed two times with 0.1 ml Hank's balanced salt solution containing 25 mM HEPES, pH 7.4, and 1 mg/ml BSA. After blotting, 0.1
10
10 -8
10 -7
Peptide (M) 125
FIG. 1. Inhibition of I-IGF I binding to UB286 (A) and SM 1.2 (B) by IGF I (•), B-chain mutant (O), [Tyr 15, Leu 16] IGF I (A), [Gin 3, Ala 4] IGF I (0) and [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I (•). Data are expressed as the percent of specific binding in the absence of competitor, and each point is the average of at least two experiments.
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IDENTIFICATION OF IGF I ANTIBODY BINDING EPITOPES
terminal residues were replaced, has only 3-fold reduced affinity for UB286 and normal affinity for SM 1.2. [Thr 49, Ser 50, He 51] IGF I and [Tyr 55, Gin 56] IGF I, two analogs in which residues in the A-region of IGF I are replaced with the analogous residues in insulin, have 30-fold and 50-fold lower affinity than IGF I for UB286 (Fig. 2). However, these two analogs and an analog in which residues 42-56 of IGF I have been replaced (A-chain mutant) have higher affinity than IGF I for SM 1.2 (Fig. 2). Other analogs in which the tyrosine residues at positions 24 and 31 were replaced ([Ser 24] IGF I and [Ala 31] IGF I), in which residues 28-37 were replaced with a four-glycine bridge ([l-27,Gly4,38-70] IGF I), or in which the eight amino acid D-region is deleted ([1-62] IGF I) have normal affinity for UB286 and SM 1.2 (Table 1). Both UB286 and SM 1.2 inhibit the binding of 125IIGF I (85 pM) to the type 1 IGF receptor in human placental membranes (Fig. 3). Half-maximal inhibition
2775
TABLE 1. Affinities of modified IGF I peptides for two antibody preparations UB286 Peptide IGF I B-chain mutant" [Gin 3, Ala 4] IGF I [Tyr 15, Leu 16] IGF I [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I [Thr 49, Ser 50, He 51] IGF I [Tyr 55, Gin 56] IGF I A-chain mutant* [Ser 24] IGF I [Ala 31] IGF I [1-27, Gly4, 38-70] IGF
IC B 0 (nM)
SM 1.2 Rel. Pot.
IC 6 0 (nM)
0.030 ± 0.008 1 2.5 ± 0.5 >2.6
Vol. 126, No. 6 Printed in U.S.A.
Identification of the Insulin-Like Growth Factor I (IGF I) Epitopes Recognized by Monoclonal and Polyclonal Antibodies to IGF I MARGARET A. CASCIERI,* MARVIN L. BAYNE, ELZBIETA BER, BARBARA G. GREEN, GINA W. MEN, AND GARY G. CHICCHI Departments of Biochemical Endocrinology and Growth Factor Research (M.L.B.), Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey, 07065
binding of 125I-IGF I to type 1 receptors on human placental membranes. In addition, SM 1.2 inhibits the ability of IGF I and IGF analogs for which it has high affinity to stimulate DNA synthesis in murine fibroblasts. In contrast, analogs with substitutions at residues 15 and 16, which have poor affinity for SM 1.2, stimulate DNA synthesis with equal potency in the presence and absence of SM 1.2. These antibodies bind normally to analogs which we have previously shown have drastically reduced binding to type 1 IGF receptors, indicating that the antibodies and the receptors recognize distinct domains of IGF I. {Endocrinology 126: 2773-2777, 1990)
ABSTRACT. We have characterized the binding epitopes of human insulin-like growth factor I (IGF I) for a polyclonal (UB286) and a monoclonal (SM 1.2) antibody using IGF analogs obtained by site-directed mutagenesis. The polyclonal antibody, UB286, which was obtained from the National Hormone and Pituitary Program, recognizes determinants surrounding residues 15 and 16 in the B-region and residues 49-51, 55 and 56 in the A-region. These residues are predicted to be within helical segments which are accessible for surface binding. The monoclonal antibody SM 1.2 selectively recognizes the region surrounding residues 15 and 16. Antibodies UB286 and SM 1.2 are both neutralizing antibodies as judged by their ability to inhibit
I
NSULIN-LIKE growth factor I (IGF I) is a polypep-
a high affinity polyclonal antibody to IGF I in 1977.
tide growth factor that has important hormonal and autocrine effects on various tissues. It is a member of a family of peptides that includes insulin and IGF II (1, 2). The amino terminal 29 amino acids of IGF I are homologous to the B-chain of insulin. The next 12 amino acids (C-region) link the B-region with the A-region (residues 42-62), which is homologous to the A-chain of insulin. The carboxyl terminus of IGF I is extended by eight amino acids to form the D-region. The biological effects of IGF I are mediated through the type 1 IGF receptor (3), although IGF I also binds to insulin receptors (4) and type 2 IGF receptors (5) with lower affinity. In addition, IGF I, but not insulin, interacts with high affinity with a family of soluble binding proteins (IGF-BP), several of which have recently been cloned (6-9). These specific binding proteins are found in serum and in the conditioned media of various cell types, and they modify IGF I activity in a complex manner (10-14). Furlanetto et al. (15) first described the preparation of
Since then, a series of polyclonal antibodies to IGF I prepared by Van Wyk and his colleagues have been widely used throughout the endocrine research community via distribution by the Pituitary Hormone Program at NIDDK. This same group has recently prepared a neutralizing monoclonal antibody to IGF I (16). At the present time, it is not known which epitopes of IGF I are involved in the binding of these antibodies to IGF I. We have used site-directed mutagenesis to determine the domains of IGF I which are involved in binding to the receptors and soluble binding proteins (17-20). The tyrosine residue at position 24 and residues between 28 and 37 in the C-region are involved in binding to the type 1 IGF receptor (17, 18). Substitutions at the amino terminus and within the first helical region of IGF I with the analogous residues in insulin selectively alter binding to the soluble binding proteins in serum (19) and in the conditioned media of various cell lines (12). Substitutions of the surface residues in the two helices in the A-region between residues 42 and 60 with the analogous residues in insulin alter binding of IGF I to the type 2 IGF receptor (20) and to IGF-BP-1 (21). In the present series of experiments, we have used these IGF analogs to map the binding epitopes for a polyclonal and a monoclonal antibody to IGF I.
Received December 11, 1989. Address all correspondence and requests for reprints to: Dr. M.A. Cascieri, Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065.
2773
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IDENTIFICATION OF IGF I ANTIBODY BINDING EPITOPES
2774
Materials and Methods Preparation of IGF analogs Analogs were prepared as previously described (17-20). Briefly, site-directed mutagenesis was performed using a synthetic gene encoding IGF I; the genes were expressed in Saccharomyces cerevisiae using a vector encoding the yeast pre-pro al protein sequence, and the secreted proteins were purified in three steps. The purity of the proteins was confirmed using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and silver staining, and the mass was determined using amino acid analysis. RIAs Antibody UB286. 125I-IGF I was prepared as previously described (22). UB286 (1:8000 final dilution) and competing peptides were incubated in 0.4 ml 0.03 M sodium phosphate, pH 7.5 containing 0.01 M EDTA, 0.02% protamine sulfate, 0.05% Tween 20, and 0.02% sodium azide. After 1 h at 22 C, 125I-IGF I (1.5 X 104 cpm) was added in 0.1 ml normal rabbit serum (1:750 final dilution). After 20 h at 4 C, 0.05 ml sheep antirabbit 7-globulin (Organon Teknika Cappel, West Chester, PA, 1:25 dilution) and 0.1 ml 20% polyethylene glycol were added. After 1 h at 22 C, samples were diluted with 1 ml assay buffer and centrifuged for 10 min at 4 C at 4000 rpm (Beckman J6B centrifuge, Beckman Instruments, Fullerton, CA). The supernatant was removed and the pellets were counted. In control experiments, normal rabbit serum (1:750) was incubated with 125 I-IGF I in the presence or absence of antibody as described above. Charcoal was added to separate free from bound ligand as described previously (17). Under these conditions, charcoal precipitation removed 70% or more than 99% of the ligand in the presence or absence of antisera, respectively. Thus, under these conditions, virtually none of the ligand bound to the small amounts of IGF-BP present in the serum.
Endo • 1990 Vol 126 • No 6
ml 0.1 N NaOH with 2% Na2CO3 was added, and cell digests were counted. Results We determined the ability of 12 analogs of IGF I to inhibit the binding of 125I-IGF I to the polyclonal antibody UB286 and to the monoclonal antibody SM 1.2. IGF I inhibits the binding to UB286 and SM 1.2 with an IC50 = 0.030 nM and 2.5 nM, respectively. The analog in which the first 16 amino acids of IGF I were replaced with the first 17 amino acids of the B-chain of insulin (B-chain mutant) has more than 100-fold lower affinity for both antibodies (Fig. 1). [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I and [Tyr 15, Leu 16] IGF I, two analogs in which residues 15 and 16 of IGF I are replaced by the analogous residues in insulin, have 25-fold and 100-fold lower affinity for UB286 and 100-fold and 25-fold lower affinity for SM 1.2, respectively (Fig. 1). In contrast, [Gin 3, Ala 4] IGF I, the analog in which only the amino
100 -
Antibody SM 1.2. Antibody was purified from ascites (Developmental Hybridoma Bank, Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, MD) using a 1-ml Protein G-sepharose 4 fast flow column (Pharmacia/LKB, Piscataway, NJ) as per manufacturer's instructions. Antibody (100 pM final concentration), 125IIGF I (1 X 104 cpm), and competing peptides were incubated in 0.5 ml 0.2 M sodium phosphate, pH 7.5, containing 0.25% BSA and 0.1% bovine 7-globulin (Pentex fraction II, ICN Immuno Biologicals, Lisle, IL). After 20 h at 4 C, added 0.5 ml 25% polyethylene glycol and centrifuged as above. DNA synthesis assays. Assays were performed in L7 murine fibroblasts as previously described (22) except for the following changes. Cells (2 x 105/\vell) were plated onto 96-well plates. After 6 h and 24 h, media were replaced with Dulbecco's modified Eagle's medium containing 0.1% calf serum. After 44 h, the media were replaced as above, and peptides and antibody were added. After 52 h, 3H thymidine (0.4 /^Ci, 25 nM) was added. After 70 h, the media were removed, cells were washed two times with 0.1 ml Hank's balanced salt solution containing 25 mM HEPES, pH 7.4, and 1 mg/ml BSA. After blotting, 0.1
10
10 -8
10 -7
Peptide (M) 125
FIG. 1. Inhibition of I-IGF I binding to UB286 (A) and SM 1.2 (B) by IGF I (•), B-chain mutant (O), [Tyr 15, Leu 16] IGF I (A), [Gin 3, Ala 4] IGF I (0) and [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I (•). Data are expressed as the percent of specific binding in the absence of competitor, and each point is the average of at least two experiments.
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IDENTIFICATION OF IGF I ANTIBODY BINDING EPITOPES
terminal residues were replaced, has only 3-fold reduced affinity for UB286 and normal affinity for SM 1.2. [Thr 49, Ser 50, He 51] IGF I and [Tyr 55, Gin 56] IGF I, two analogs in which residues in the A-region of IGF I are replaced with the analogous residues in insulin, have 30-fold and 50-fold lower affinity than IGF I for UB286 (Fig. 2). However, these two analogs and an analog in which residues 42-56 of IGF I have been replaced (A-chain mutant) have higher affinity than IGF I for SM 1.2 (Fig. 2). Other analogs in which the tyrosine residues at positions 24 and 31 were replaced ([Ser 24] IGF I and [Ala 31] IGF I), in which residues 28-37 were replaced with a four-glycine bridge ([l-27,Gly4,38-70] IGF I), or in which the eight amino acid D-region is deleted ([1-62] IGF I) have normal affinity for UB286 and SM 1.2 (Table 1). Both UB286 and SM 1.2 inhibit the binding of 125IIGF I (85 pM) to the type 1 IGF receptor in human placental membranes (Fig. 3). Half-maximal inhibition
2775
TABLE 1. Affinities of modified IGF I peptides for two antibody preparations UB286 Peptide IGF I B-chain mutant" [Gin 3, Ala 4] IGF I [Tyr 15, Leu 16] IGF I [Gin 3, Ala 4, Tyr 15, Leu 16] IGF I [Thr 49, Ser 50, He 51] IGF I [Tyr 55, Gin 56] IGF I A-chain mutant* [Ser 24] IGF I [Ala 31] IGF I [1-27, Gly4, 38-70] IGF
IC B 0 (nM)
SM 1.2 Rel. Pot.
IC 6 0 (nM)
0.030 ± 0.008 1 2.5 ± 0.5 >2.6
