Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding

Jim Xiang1, Lata Prasad2, Louis T.J. Delbaere2 and Zongchao Jia3

Saskatoon Cancer Center, Departments of Oncology and 2 Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 4H4 and 3 Department of Biochemistry, Queen's University, Kingston,Ontario K7L 3N6, Canada


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Keywords: binding affinity/FR residues/site-directed mutagenesis/TAG72 antigen


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The determination of the three-dimensional structures of antibody fragments by X-ray crystallography has led to the realization that the polypeptide chains of the immunoglobulin G (IgG) molecules are folded into globular domains, four in the heavy (H) and two in the light (L) chain, which are connected by extended peptide segments like pearls on a string (Amzel and Poljak, 1979Go). All domains exhibit a similar polypeptide folding pattern characterized by two ß-sheets. Sequence comparisons among heavy- and light-chain variable (V) domains reveal that each domain has three complementary-determining regions (CDR) flanked by four framework regions (FR) of less variable sequences (Kabat et al., 1991Go). The antibody-combining site is formed by the juxtaposition of six CDRs appearing as loops at one end of the ß-sheet, three from VH and another three from VL. These CDR loops present a surface that interacts with the antigen and determine the antibody specificity and the antigen binding affinity. Although the FRs comprise the conserved ß-sheet framework and are involved in the interchain interactions that bring domains together (Chothia et al., 1985Go), some of them may also directly or indirectly contribute to the antigen binding. For example, some FR residues, especially those in conjunction with CDR, have been found on occasion to be involved in the direct interaction with the antigen (Fischmann et al., 1991Go; Tulip et al., 1992Go). Sometimes, FR residues having the atomic interaction with CDR residues were found to influence indirectly the antibody binding affinity by altering the conformation of CDR loops (Kettleborough et al., 1991Go; Foote and Winter, 1992Go; Lavoie et al., 1992Go; Kao and Sharon, 1993Go; Xiang et al., 1995Go).

B72.3 is a mouse antibody with specificity for the tumor-associated TAG72 antigen (Thor et al., 1986Go). The TAG72 epitope defined by the B72.3 antibody is NeuAc2–6{alpha}GalNAc{alpha}1-O-Ser/Thr (sTn) (Kjeldsen et al., 1988Go). Recently, the data showed that the minimal epitope for the B72.3 antibody is the dimeric sTn–serine cluster (sTn2) (Reddish et al., 1997Go). The crystallization of the chimeric B72.3 Fab' fragment was reported previously (Brady et al., 1991Go). A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 Å with an R-factor of 17.6%. The crystallographic analysis of the chimeric B72.3 Fab' illustrated that some H-chain FR residues (H71, H73 and H93) form hydrogen bonds to CDR residues (Brady et al., 1992Go). These H-chain FR residues may affect the conformation of H-chain CDR loops. Our site-directed mutagenesis study (Xiang et al., 1995Go) has confirmed that H-chain FR residues 71 and 93 are the major determinants for H-chain CDR loop conformations. A single amino acid substitution at these FR residues significantly reduced the binding affinity for the TAG72 antigen by 12- and 20-fold, respectively. In addition to atomic interactions between H-chain FR and CDR residues, the crystallographic study has also illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain, although there are many main-chain atoms of FR residues that make hydrogen bonds to CDR residues. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the carbonyl group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of hydrogen bonds at these FR residues may affect L-chain CDR loop conformations. To confirm these assumptions, we conducted site-directed mutagenesis at these mouse FR residues. The binding affinities of these mutant antibodies were measured in a solid-phase radioimmunoassay (RIA) and compared with the original chimeric antibody cB72.3-1-3 (Xiang et al., 1990Go).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Expression vector, antigen and cell line

The expression vectors mpSV2neo-EP-VH-C{gamma}1 and mpSV2gpt-EP-C{kappa} were previously constructed for expression of the cB72.3-1-3 H- and L-chains, respectively (Xiang et al., 1990Go, 1992Go). Bovine mucin from submaxillary glands (Sigma Chemical, St Louis, MO) containing the TAG72 epitope (Xiang et al., 1990Go) was used as the antigen source. The SP2/0Ag14 myeloma cell line lacking the expression of its own internal H- and L-chains was obtained from the American Type Culture Collection (ATCC, Rockville, MD). This cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).

Site-directed mutagenesis

Amino acids of the cB72.3-1-3 V{kappa} region are numbered according to Kabat's method (Kabat et al., 1991Go) and shown in Figure 1Go. The major atomic interactions between L-chain FR-CDR residues are illustrated in Figure 2Go. To eliminate the hydrogen bond (4.7 Å) between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1 (Figure 2AGo), oligo 29 (5' gacat ccaga tggct cagtc tccag 3') was synthesized for site-directed mutagenesis. It is complementary to L-chain FR1 with substitution of threonine by alanine at L5. To eliminate the hydrogen bond (2.61 Å) between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 (Figure 2BGo), oligo 30 (5' aattt agcat ggttt caaca gaaac 3') was synthesized for site-directed mutagenesis. It is complementary to the L-chain FR2 with substitution of tyrosine by phenylalanine at L36. To eliminate the hydrogen bonds (2.65 and 3.02 Å) between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 and also the amide nitrogen group of serine at L31 in CDR1 (Figure 2CGo), oligo 31 (5' cggca cacag ttttc cctca agatc 3') complementary to the L-chain FR3 was synthesized for site-directed mutagenesis. Mutations were introduced into M13mp18-V{kappa} by these three primers in site-directed mutagenesis to form three plasmids M13mp18-V{kappa}M29–31. Sequences of these mutant V{kappa}M29–31 regions were verified by the dideoxy nucleotide sequencing method (Sanger et al., 1977Go).



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Fig. 1. Amino acid sequence of the cB72.3 V{kappa} region. The one-letter amino acid code is used. Amino acids are numbered sequentially according to Kabat's method. Demarcated are respective framework regions (FR), complementary-determining regions (CDR) and joining segment (J).

 


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Fig. 2. Stereo diagrams of some L-chain FR residues with atomic interaction with L-chain CDR residues from the coordinates of crystallographic analysis of chimeric B72.3 Fab' deposited in the Brookhaven Data Bank. (A) The hydrogen bond formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1. (B) The hydrogen bond formed between the hydroxyl group of tyrosine at L36 and the amide nitrogen group of glutamine at L89 in CDR3. (C) The hydrogen bond formed between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1 The {alpha}-carbon trace and the amino acid side-chain conformation are marked. Putative hydrogen bonds are shown as broken lines.

 
Construction of expression vectors

The expression vectors mpSV2gpt-EP-V{kappa}M29–31-C{kappa} were constructed for production of mutant chimeric L-chains. The mutant V{kappa}M29–31 region cDNA fragments (KpnI) was purified from M13-mp18-V{kappa}M29–31 by KpnI digestion and introduced into the KpnI site in the multiple cloning region of mpSV2gpt-EP-C{kappa} (Xiang et al., 1992Go) to form the mutant L-chain expression vectors mpSV2gpt-EP-V{kappa}M29–31-C{kappa}. These expression vectors contain the gpt gene for mycophenolic acid selection and a complete transcription unit including enhancer (E), immunoglobulin promoter (P), mutant V{kappa}M29–31 region cDNA fragments and human genomic DNA fragment of {kappa} constant region (C{kappa}).

Expression and purification of mutant chimeric antibodies

The mutant L-chain expression vector DNA mpSV2gpt-EP-V{kappa}M29–31-C{kappa} was first transfected into SP2/0Ag14 cells by electroporation as described (Xiang et al., 1990Go). Cells were selected for growth in media containing mycophenolic acid at 0.8 µg/ml. After 14 days, growth supernatants were screened in a human L-chain capture ELISA for examining the expression of mutant chimeric L-chains (Xiang et al., 1996Go). The positive clones derived from SP2/0Ag14 cell line secreting mutant chimeric L-chains were further transfected with the chimeric H-chain expression vector DNA mpSV2neo-EP-VH-C{gamma}1 (Xiang et al., 1990Go) and selected for growth in media containing both G418 at 2 mg/ml and mycophenolic acid at 0.8 µg/ml. The growth supernatants were then screened by a TAG72-binding ELISA for examining the expression of three mutant chimeric antibodies cB72.3m29–31 (Xiang et al., 1990Go). These mutant chimeric antibodies were further purified from supernatants by protein A-Sepharose chromatography (Xiang et al., 1990Go). Protein concentrations were determined using a Bio-Rad (Richmond, CA) protein assay kit according to the method described in the manual.

Affinity constants

The affinity constants (Ka) of mutant chimeric antibodies cB72.3m29–31 were determined in a solid-phase RIA using the bovine mucin as a source of the TAG72 antigen as described previously (Xiang et al., 1993Go). Briefly, serial dilutions of mutant antibodies were added to each mucin-coated well (in triplicate) of the first microtiter plate for incubation overnight at 4°C. The supernatants of each well, which contained the free mutant antibody, were transferred to each well of the second microtiter plate, which had previously been coated with goat anti-human IgG antibody. The amount of bound and free antibodies on the first and second plates was measured using the 125I-labeled goat anti-human IgG antibody. To calculate Ka, the method of Scatchard (1949) was used. The ratios of the concentrations of bound to free antibody were plotted against the concentration of bound antibody. The slope represents Ka of each mutant antibody (Xiang et al., 1993Go).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The FRs of antibodies fold into a conserved ß-sheet structure that acts as scaffolding for the antigen-contacting CDR. Some FR residues may also directly affect the conformation of CDR loops by atomic interactions between FR and CDR residues (Kettleborough et al., 1991Go; Foote and Winter, 1992Go; Lavoie et al., 1992Go; Xiang et al., 1995Go). According to the crystallographic study of chimeric B72.3 Fab', there are hydrogen bonds formed between side-chains of three L-chain FR residues (L5, L36 and L71) and CDR residues (Figure 2A–CGo). These three FR residues may thus affect the conformation of L-chain CDR loops. To confirm these assumptions, we performed site-directed mutagenesis at these three FR positions (L5, L36 and L71) with three oligonucleotides (oligos 29–31) resulting in three mutant chimeric antibodies cB72.3m29–31. The affinity constants of these three mutant antibodies for the TAG72 antigen were determined in a solid-phase RIA compared with that of the original cB72.3-1-3 antibody.

The hydrogen bond formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1 is a borderline one because of the long distance of 4.17 Å. It would be a very weak interaction, if it could indeed be considered as a hydrogen bond at all. To clarify the ambiguity and determine whether it is an important interaction, we performed a single amino acid substitution of threonine by alanine at L5 to eliminate this hydrogen bond. Our results showed that the affinity constant (Ka) of cB72.3m29 with substitution of threonine by alanine at L5 is 6.5x 108 M–1, which is similar to that of the original cB72.3-1-3 antibody (6.8x108 M–1) (Table IGo), indicating that this weak interaction is not important. This is not surprising in the light of the long interaction distance of 4.17 Å. The influence of threonine at L5 on the CDR loop conformation would be minimal. Arginine at L24 of CDR1, being surface exposed, is very flexible and one would not expect that a weak interaction with its guanidinal group could exert much effect on the main-chain conformation. This notion is evidently supported by our mutagenesis result. Alternatively, it could be that residues and solvents rearranged to compensate for the missing hydrogen bond caused by the site-directed mutagenesis. Therefore, it does not play any role in the L-chain CDR1 loop conformation. In addition, a strong hydrogen bond is formed between the main-chain carbonyl groups of isoleucine at L2 in FR1 and serine at L26 in CDR1 (Brady et al., 1992Go), which may play some role in keeping the L-chain CDR1 loop conformation.


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Table I. Affinity constants of mutant cB72.3m antibodies
 
A single amino acid substitution of tyrosine by phenylalanine at L36 and L71 eliminated the hydrogen bonds formed between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3, and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Our results showed that the affinity constant (Ka) of cB72.3m30 with substitution of tyrosine by phenylalanine at L36 is 7.0x108 M–1, which is similar to that of the original cB72.3-1-3 antibody (6.8x108 M–1), while the Ka of cB72.3m31 (0.3x108 M–1), with substitution of tyrosine by phenylalanine at L71, was significantly reduced by 23-fold (Table IGo), indicating that L71 in FR3 plays an important role in influencing the TAG72 antigen binding. Interestingly, the importance of the L71 residue in the CDR loop conformation has also been reported previously in an anti-lysozyme D1.3 antibody (Foote and Winter, 1992Go). Substitution of phenylalanine by tyrosine at L71 of D1.3 antibody resulted in an additional contribution of 0.8 kcal/mol in binding affinity for the lysozyme.

It is not totally surprising to see that the mutation of tyrosine to phenylalanine at L36 did not affect antigen binding. Generally, side-chain interactions would not necessarily affect the main-chain conformation. Even if they do, they would be less effective than interactions that directly involve main-chain atoms. Akin to arginine at L24, glutamine at L89 has a relatively long side-chain, and loss of a hydrogen bond may just increase its side-chain flexibility which does not have much impact on the main-chain conformation. As far as tyrosine at L71 is concerned, the interactions are much more significant. First, there are two hydrogen bonds with distances of 2.65 and 3.02 Å. More importantly, these two hydrogen bonds involve main-chain atoms of isoleucine at L29 and serine at L31. The hydroxyl group of tyrosine at L71 does not make any hydrogen bond to side-chain atoms. Loss of these two hydrogen bonds decreased its antigen binding by 23-fold, strongly suggesting the critical role that tyrosine at L71 plays. In this case, it is plausible that the main-chain interactions have an important structural role and loss of these two critical main-chain hydrogen bonds could translate into a conformational change in and around the isoleucine (L29)–serine (L31) segment of CDR. Alternatively, it could be due to an entropic effect residing in the overall tightening upon the antigen binding of a structure that was rendered more mobile by the amino acid replacement at L71, without really affecting the CDR loop conformation. Without structural data, it is indeed very difficult to ascertain whether these hydrogen bonds could influence the CDR loop conformation. However, based on the mutagenesis, binding affinities and structural comparison involving L5, L36 and L71, in which only main-chain interactions seem to have a significant impact, we suggest that tyrosine at L71 is an important residue that is involved in the stabilization of the CDR loop conformation. It is evident that CDR main-chain hydrogen bonds are important and may have a general implication when humanization of B72.3 antibody is considered.

A number of mouse/human chimeric B72.3 antibodies have been constructed by recombinant DNA technology in order to reduce the human anti-mouse antibody response (Whittle et al., 1987Go; Xiang et al., 1992Go). However, clinical studies suggested that an anti-idiotypic immune response to the V region was still present (Khazaeli et al., 1991Go). To minimize the anti-idiotype response, the approach of genetic engineering of a humanized antibody has been taken, in which mouse CDR loops will be directly grafted on to a human framework (Jones et al., 1986Go). To retain the binding specificity and affinity of its mouse counterpart, some important mouse FR residues were maintained in humanized antibodies (Graziano et al., 1995Go; Presta et al., 1997Go). For construction of a humanized B72.3 antibody, a proper human framework with the highest homology to the B72.3 antibody should be chosen for maintenance of B72.3 CDR loop conformations. Based on the sequence homology search using the MicroGenie sequence analysis software of Beckmann, the human myeloma protein Eu showed the highest homology to both the B72.3 VH (59%) and V{kappa} (63%) regions simultaneously (Glaser et al., 1992Go). In this paper we have reported our experimental proof of the importance of the L-chain FR residue (L71) in influencing the TAG72 antigen binding. However, the L-chain FR residue of Eu at L71 is phenylalanine. Therefore, our results will be of importance when the humanized B72.3 antibody is constructed by grafting the B72.3 CDRs on to the Eu FRs, since this important mouse FR residue tyrosine at L71 must be maintained in the final humanized B72.3 antibody.


    Acknowledgments
 
This study was supported by a research grant funded by the Saskatchewan Cancer Agency.


    Notes
 
1 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received August 7, 1998; revised November 18, 1998; accepted February 10, 1999.