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The Journal of Immunology, 1998, 160: 4406-4417.
Copyright © 1998 by The American Association of Immunologists

Only Selected Light Chains Combine with a Given Heavy Chain to Confer Specificity for a Model Glycopeptide Antigen1

Marcin Czerwinski*, Dorota Siemaszko*, Don L. Siegel{dagger} and Steven L. Spitalnik2,{dagger}

* Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland; and {dagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Determining the rules governing the formation of a light chain:heavy chain pair with a given Ag specificity is important for understanding Ab structure and function. In particular, differing results concerning the issue of heavy (or light) chain promiscuity have been obtained. For example, evidence for heavy chain promiscuity was clearly identified for human Abs recognizing HIV gp120 (1). In contrast, other groups found that heavy chain:light chain pairing is far from random, and that Ag specificity is maintained only if very similar light chains are interchanged with a given heavy chain (2, 3). It is also not clear whether the chemical nature of the epitope (i.e., peptide, oligosaccharide, hapten, glycopeptide, etc.) recognized by the Ab is involved in determining the promiscuity of pairing of the corresponding heavy (or light) chain.

Few studies have examined the humoral immune response to glycopeptide Ags (4, 5, 6). To this end, we studied Abs recognizing the clinically relevant M and N human blood group Ags that are carried by glycophorin A, an RBC membrane glycoprotein (7, 8). The M and N Ags are defined by amino acid polymorphisms at positions 1 and 5 of glycophorin A (9, 10, 11, 12, 13):

1 2 3 4 5 M: NH2 Ser Ser Thr Thr Gly R N: NH2 Leu Ser Thr Thr Glu R

The serine and threonines at positions 2 to 4 are each glycosylated with sialylated O-glycans (9). Many M- and N-specific mouse mAbs have been characterized (14, 15, 16, 17, 18, 19, 20, 21). Most recognize complex glycopeptide epitopes that depend on both the amino acid polymorphisms and the intact O-glycans (4, 17, 18, 19, 21, 22, 23).

We previously showed that the murine immune response against the N, but not the M, Ag may be restricted (24). The heavy chains of four anti-N mouse mAbs that were obtained from different fusions each contained a VH region derived from the VH2 (Q52) germ-line gene family, and each used the same JH4 gene segment. In addition, two of the anti-N light chains used Vk regions derived from the Vk8 germ-line gene family and the same Jk1 gene segment. However, the remaining two mAbs used light chain V regions derived from other Vk germ-line gene families. This suggested that the apparent restriction of the immune response may be limited to the use of particular highly homologous heavy chains that might be able to combine promiscuously with various light chains to yield anti-N specificity.

To investigate this apparent heavy chain restriction further, the Fab of various anti-N (and anti-M) mouse mAbs were expressed as Fab phage (25). The fine specificity of each of these Fab phage, such as their dependence on either sialylation of the target Ag or pH, was similar to that of the corresponding parental mAb. Light chain shuffling was used to examine whether various light chains from anti-N mAbs could couple with particular anti-N Fd to yield N-specific Abs. Although each shuffling experiment yielded an intact Fab, none recognized any epitope on glycophorin A.

Since the previous limited light chain shuffling experiments suggested the importance of particular heavy chain:light chain pairing rather than heavy chain promiscuity (25), we decided to study this further by coupling a wide array of light chains to single anti-N (and anti-M) heavy chains. This allows a direct examination of the flexibility of pairing of a given heavy chain with various light chains to yield specific binding to these glycopeptide Ags. To this end, light chain libraries obtained from the mice immunized with glycophorin A were coupled to Fd derived from specific anti-M and anti-N mouse mAbs. After expression as Fab phage followed by panning on glycophorin A, Ag-binding clones were evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Glycophorin A

Glycophorin A of the M or N blood type was purified from human RBCs by phenol extraction (16). Purified glycophorin A was 125I labeled (26) using Iodo-Gen (Pierce, Rockford, IL).

Mouse mAbs

The 425/2B, N61, and N92 mouse mAbs, which recognize epitopes on glycophorin A (16, 21), were isolated and characterized previously (see Table IGo). The anti-M and anti-N mAbs were purified from culture supernatant using affinity chromatography on protein G-agarose (Boehringer Mannheim, Mannheim, Germany). The bound IgG fraction was eluted with 100 mM glycine-HCl, pH 2.8, and immediately neutralized with 1 M Tris, pH 8.0. After dialysis against Tris-buffered saline (TBS3; 20 mM Tris-HCl, pH 7.5 or 8.3, and 150 mM NaCl) and concentration by ultrafiltration, the protein content was measured (27) using BSA as a standard. The purity of these fractions was evaluated by SDS-PAGE, as described below. As a control, 4/3/17, an IgG mouse mAb that recognizes an epitope on carcinoembryonic Ag (28), was obtained from Dr. Fritz Grunert (Freiburg, Germany) and purified as described above.


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  		Table I. Glycophorin A-specific mouse mAbs

 
Monoclonal Fab phage

The parental Fab phage used in this study were derived from the hybridoma mAbs described in Table IGo. These Fab phage were constructed previously using the pComb3 vector (29), and their sequences were determined (24). For the current study, each mAb-derived Fd and its corresponding light chain were inserted into the pComb3H vector (30) using standard techniques (see below). The pComb3H vector was obtained from the Scripps Research Institute (La Jolla, CA).

Mouse immunization and library construction

Male BALB/c mice (6 wk old) were immunized i.p. with 10 µg of purified M- or N-type human glycophorin A in CFA. The immunization was repeated after 2 wk in IFA. One week later, total splenic RNA was prepared (31). Following reverse transcription using the manufacturer’s directions for the Superscript II enzyme (Life Technologies, Gaithersburg, MD), complete light chain cDNAs were amplified by PCR, using the amplification conditions and primers described previously (25, 32). An additional 5' primer based on a published sequence (33) was also used: 5'-CC(A/G)(A/T)T(C/G)(C/G)GAGCTC(A/C)AGAT(A/G)A(C/T)CCAG (A/T)CT(A/C)CA-3'.

The oligonucleotide primers were synthesized at the Wistar Institute (Philadelphia, PA) and purified by HPLC. Amplified light chain cDNA was then gel purified, digested with both XbaI and SacI (Boehringer Mannheim, Indianapolis, IN), gel purified again, and ligated into modified pComb3H vectors, which were previously digested with the same enzymes. The pComb3H vectors used for this purpose had already been modified to contain cDNAs encoding the heavy chain V regions from one of the following mouse mAbs (see Table IGo): N61 (anti-N), N92 (anti-N), or 425/2B (anti-M). Therefore, in each case this approach would produce a library of phagemids containing one particular heavy chain V region and any of a large number of light chains.

The ligation product was introduced into Escherichia coli (XL-1 Blue, Stratagene, La Jolla, CA) by electroporation, and the culture was grown overnight in medium containing 100 µg/ml carbenicillin and 10 µg/ml tetracycline. The size of the light chain library was determined by plating several dilutions of an aliquot of the medium after electroporation. Phagemid DNA containing the light chain library was prepared from the overnight culture using Qiagen columns (Chatsworth, CA). The presence of light chain inserts in individual, randomly selected colonies was verified as previously described (29). Plasmids containing inserts were sequenced by the dideoxy method using a Sequenase 2.0 sequencing kit (U.S. Biochemical, Cleveland, OH) and the universal 5' primer, 5'-AAAGACAGCTATCGCGATTG-3'. The reverse, antisense primer, 5'-GCACACGACTGAGGCACCTCC-3', which is complementary to codons 127 through 134 of the light chain C region, was used to sequence the 3' portion of the V region of the light chain.

The pComb3H vector containing a given monoclonal heavy chain Fd and the light chain library was then introduced by electroporation into E. coli (SURE strain, Stratagene). The bacteria were grown at 37°C to an OD600 of 1.0 in 20 ml of Super Broth medium (30 g/l tryptone, 20 g/l yeast extract, and 10 g/1 3-(N-morpholino)propanesulfonic acid (pH7.0)) containing 100 µg/ml carbenicillin and 10 µg/ml tetracycline. At that time, 1012 plaque-forming units of VCSM13 helper phage (Stratagene) were added. The bacteria were then incubated at 37°C for 15 min, concentrated by centrifugation, and resuspended in 100 ml of fresh Super Broth medium containing antibiotics as described above. The culture was incubated at 30°C overnight, the supernatant was cleared by centrifugation, and the Fab phage were precipitated with polyethylene glycol, as described previously (29). The resulting Fab phage were resuspended in 1 ml of PBS (0.01 M Na2HPO4/NaH2PO4 and 0.15 M NaCl, pH 7.4) and quantified by titration of CFU (29).

Panning of the combinatorial library

Panning was performed using a modification of a previously described procedure (29). In brief, four wells of a 96-well microtiter plate (Immulon 2, Dynatech, Alexandria, VA) were coated overnight at 4°C with 100 µl of 20 µg/ml of purified glycophorin A in 50 mM carbonate buffer, pH 9.6. The wells were washed twice with distilled water and blocked by completely filling the well with PBS containing 3% (w/v) BSA and then incubating the plate at room temperature for 1 h. Following removal of the blocking solution, 50 µl of the phage library (typically 1010 plaque-forming units (pfu)) were added to each well, and the plate was incubated for 2 h at room temperature. Phage were then removed, and the plate was washed 10 times with TBS over a period of 1 h at room temperature. To elute the bound phage, the wells were filled with 100 µl of TBS, and the plate was incubated at 60°C for 8 min. The eluted phage were used to infect 2 ml of fresh E. coli SURE cells, previously grown to an OD600 of 1, for 15 min at room temperature, after which 10 ml of Super Broth medium containing 20 µg/ml carbenicillin and 10 µg/ml tetracycline were added. Aliquots were removed for plating to determine the number of phage that eluted from the microtiter plate wells. The culture was shaken for 1 h at 37°C, after which it was added to 100 ml of Super Broth containing 100 µg/ml carbenicillin and 10 µg/ml tetracycline and shaken for an additional hour. VCSM13 helper phage (1012 pfu) were added, and following an overnight incubation at 30°C, the supernatant was cleared by centrifugation, and the phage were prepared, as described above.

In an alternative approach, human RBCs were used for panning. Human RBCs were collected in the presence of EDTA and then typed for the presence of the M and N Ags using standard serologic methods. For panning, 1010 pfu of a phage library were added to 10 µl of packed RBCs (heterozygous MN blood type) in a total volume of 200 µl of TBS (at either pH 7.4 or 8.3). Following a 2-h incubation at room temperature on a laboratory rotator, the RBCs were pelleted by centrifugation at 10,000 x g for 30 s and washed five times with 1 ml of TBS. The bound phage were eluted by incubation of the RBCs at 60°C for 8 min. The eluted phage were then used to infect E. coli SURE cells, as described above.

Colony screening of panned libraries

Bacteria obtained from each round of panning were streaked on Super Broth agar plates containing 100 µg/ml carbenicillin and 10 µg/ml tetracycline and cultured at 37°C for 4 h. The plates were then overlaid with nitrocellulose filters (82 mm in diameter) soaked in 5 mM isopropyl-D-thiogalactopyranoside (Roth, Karlsruhe, Germany). Following an overnight incubation at 30°C, the filters were removed, incubated in a chloroform chamber for 15 min, transferred to 25 ml of lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl2 containing 3% BSA, 40 mg of lysozyme, and 100 U of DNase), and rocked for 2 h. Filters were also blocked in TBS containing 4% BSA for 1 h at room temperature. Filters were then probed with Ag by incubation for 2 h at room temperature in TBS (10 ml/filter) containing 2% BSA and 10 µg of 125I-labeled glycophorin A (M or N type). Filters were washed 10 times in TBS, dried, and exposed to x-ray film. Colonies that yielded positive signals were expanded, the resulting plasmids were purified using standard methods (34), and the light chain cDNA inserts were sequenced, as described above. Alternatively, the colonies were expanded, and phage were prepared, as described above.

Preparation of soluble Fab from Fab phage

The pComb3H vectors containing the heavy and light chain cDNAs of interest were digested with NheI and SpeI to remove the gene III fragment, gel purified, and dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim). A double-stranded synthetic oligonucleotide encoding six histidine residues, a C-terminal glycine, and a stop codon and containing an NcoI restriction site (35) was inserted into the resulting SpeI/NheI site of the digested pComb3H vectors. The orientation of the insert was confirmed by nucleotide sequencing by the dideoxy method using the following reverse antisense primer: 5'-TTTGCCGATTTCGGCCTATTGG-3'.

The resulting vectors encoding soluble Fab were electroporated into E. coli SURE cells, and the bacteria were plated. Bacteria from single colonies were each grown at 37°C overnight in 10 ml of Super Broth medium supplemented with 100 µg/ml ampicillin and 10 µg/ml tetracycline. The next day, 500-µl aliquots from these cultures were used to inoculate 100 ml of Super Broth medium containing 100 µg/ml ampicillin, 10 µg/ml tetracycline, 0.1% glucose, and 20 mM MgCl2. The cultures were grown at 37°C until they reached an OD600 of 0.2, and isopropyl-D-thiogalactopyranoside was added to a final concentration of 1 mM. Following additional growth at 22°C for 12 to 16 h, the bacteria were collected by centrifugation, and the periplasmic proteins were released by osmotic shock (36). In brief, the bacteria were incubated in 50 ml of 20% sucrose/30 mM Tris, pH 8.0, for 20 min, centrifuged, and resuspended in 50 ml of 5 mM MgSO4. Following centrifugation, both supernatant fractions were combined and dialyzed against 50 mM Na2HPO4/NaH2PO4 and 300 mM NaCl, pH 8.0.

A column packed with 0.6 ml of nickel-NTA-agarose (Qiagen) was first washed with 50 mM Na2HPO4/NaH2PO4 and 300 mM NaCl, pH 8.0, and then loaded with the periplasmic fraction of proteins obtained as described above. The column was washed with 50 mM Na2HPO4/NaH2PO4 and 300 mM NaCl, pH 6.5, until the OD280 reverted to baseline. The column was then washed with 10 and 20 mM imidazole in 50 mM Na2HPO4/NaH2PO4 and 300 mM NaCl, pH 6.5, buffer, and the Fab were eluted with 50 mM imidazole in this buffer. After dialysis against TBS and concentration by ultrafiltration, the protein content was measured, and the purity was evaluated by SDS-PAGE, as described below.

Microplate ELISA

Microtiter plates (Immulon 2, Dynatech) were coated either with glycophorin A purified from human RBCs (20 µg/ml in 50 mM carbonate buffer, pH 9.6), as described above, or with goat anti-mouse IgG (Sigma) diluted 1/100 in PBS. Nonspecific binding was blocked by incubation for 1 h at room temperature with PBS containing 3% BSA. After washing the wells once with distilled water, Fab phage appropriately diluted in TBS (at pH 7.4 or 8.3) were added, and the mixture was incubated for 1 h at room temperature. After five washes with TBS, the bound Fab phage were detected with biotinylated sheep anti-M13 Ab (5 Prime -> 3 Prime, Boulder, CO) diluted 1/1000 in TBS. Peroxidase-conjugated streptavidin (Life Technologies) diluted 1/1000 in TBS and o-phenylenediamine (Sigma) were used to develop the reaction. The OD490 of each well was then determined with an EL 311 spectrophotometer (Behring, Marburg, Germany).

The binding of purified soluble Fab or purified mAbs was evaluated similarly to that described above, but rabbit anti-Fab Ab conjugated with peroxidase (Pierce) was used as a secondary reagent.

Electrophoresis and Western blotting

The proteins were separated by SDS-PAGE (37) using 10% gels and stained with Coomassie Brilliant Blue (Sigma). Alternatively, the electrophoretically separated proteins were transferred to nitrocellulose (38). The blots were blocked for 1 h with 2% BSA in PBS and overlaid either with the appropriately diluted intact Ab or with the Fab. Following a 1-h incubation at room temperature, the blots were washed with TBS and overlaid with a 1/1000 dilution of rabbit anti-mouse Fab Ab conjugated with alkaline phosphatase (Pierce). Following a 1-h incubation at room temperature, the blot was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of the light chain libraries

We previously used phage display technology to express the Fab of several anti-glycophorin A mAbs (25). Three mAbs from that group, N61, N92, and 425/2B, were also used in the current study (Table IGo). Although these monoclonal Fab phage were previously characterized in detail (25), they were expressed in the pComb3 vector. We subsequently found that the pComb3 vector caused persistent problems with homologous recombination and deletions during panning experiments (data not shown). Therefore, the same Fab were expressed in a new, modified vector, pComb3H, which lacks repetitive sequences (30), and thus is stable during bacterial culture. The immunologic characteristics of the N61, N92, and 425/2B Fab phage expressed by the pComb3H vector were very similar to those obtained previously with the pComb3 vector (data not shown). Therefore, all the experiments described in the current communication used the pComb3H vector.

We previously found that phage displaying chimeric Fab, consisting, for example, of the Fd from the N61 mAb and the light chain derived from several other N-specific hybridoma mAbs, did not bind glycophorin A (25). To examine this issue further, mice were immunized with M- or N-type glycophorin A. Immunized mice were used in an effort to increase the chances of finding heavy chain:light chain pairs with the desired Ag specificity (32, 39). To prepare light chain libraries from individual mice, total cDNA was isolated from splenocytes, and then light chain cDNA was amplified by PCR. The light chain libraries obtained from spleens of individual mice immunized with M-type glycophorin A were ligated into the pComb3H phagemid containing the Fd of the anti-M mAb 425/2B. Similarly, the light chain libraries obtained from spleens of individual mice immunized with N-type glycophorin A were ligated into a pComb3H phagemids containing the Fd of one of either of the two anti-N mAbs, N61 and N92.

Analysis of clones from the unpanned Fd:light chain libraries

From each of the resulting, unpanned monoclonal Fd:light chain libraries, five to nine clones were randomly chosen and expanded, and their light chain sequences were determined. Of the 20 clones examined, no two clones were found with identical light chain V regions. Typically, approximately 50% of the light chains belonged to the Vk4 and Vk5 subgroups (Table IIGo), which comprise the largest Vk gene families in the mouse, consisting of 25 to 50 germ-line genes (40). Light chain cDNAs belonging to the Vk8, Vk11, Vk12/13, and Vk19/28 families were also found, suggesting that the light chain libraries contained a representative and diverse population of mouse light chains. In addition, sequences corresponding to the Jk1, Jk2, Jk4, and Jk5 gene segments were found in the unpanned light chain cDNA library. None of the eight randomly selected unpanned Fab phage that were tested were found to bind to M- or N-type glycophorin A.


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  		Table II. Light chain sequences of randomly isolated clones from the unpanned Fd fragment:light chain libraries

 
Analysis of clones from the panned Fab phage:light chain libraries

The size and the Ag-specific enrichment of the light chain libraries after each panning step are presented in Table IIIGo. Bacterial colonies representing from 3 to 45% of Fab phage after the first round of panning were detected using 125I-labeled glycophorin A. After a second round of panning, that ratio increased to 22 to 65%. Clones that were positive in the 125I-labeled glycophorin A binding assay were randomly selected and expanded, their light chain sequences were determined, and their immunologic characteristics were evaluated. The numbers of clones from each round of panning that were sequenced and evaluated are shown in Table IIIGo. In selected cases the Fd was also resequenced to ensure that no mutations were introduced in the construction of these Fab phage libraries.


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  		Table III. Panning results for the Fd fragment:light chain libraries

 
Chimeras isolated from the 425/2B Fd:anti-M light chain libraries

Sequence information. When light chain libraries obtained from two mice immunized with M-type glycophorin A were coupled to the 425/2B Fd, and the resulting 425/2B Fd:anti-M light chain libraries were panned on M-type glycophorin A, three different types of clones reacting with M-type glycophorin A were identified. These are represented by AP1, A4, and DB1. DB1 was obtained from the M1 light chain library; AP1 and A4 were obtained from the M3 library (Table IIIGo). The complete nucleotide sequences of these light chain V regions are shown in Figure 1Go. In contrast, no positive clones were found after panning the 425/2B Fd:anti-M light chain libraries on N-type glycophorin A. The AP1, A4, and DB1 clones all used the same VkRF germ-line gene as the parental hybridoma Ab, 425/2B. Since no germ-line gene belonging to the VkRF gene family has been published, the sequence of an anti-influenza virus hemagglutinin Ab light chain (H37-90) (41), which revealed the greatest homology with the 425/2B light chain, is shown for comparison. The amino acid sequence homology between the 425/2B light chain V region and those of AP1, A4, and DB1 is approximately 92%. The amino acid differences between the new clones and the 425/2B light chain at codons 1 to 5 are due to differences in the oligonucleotide primers used for amplification, which, based on their synthesis in vitro, determine the sequences of codons 1 through 8. Interestingly, the new clones used the Jk2 gene segment, whereas the Jk5 gene segment was found in the 425/2B light chain. As can be seen from comparison of the 425/2B light chain with the library-derived light chains, there are multiple positions at which the new clones have a different amino acid sequence from the 425/2B light chain, but are nonetheless identical with the anti-hemagglutinin light chain (e.g., at codons 10, 28, 43, 77, and 93). One of these substitutions is in CDR1 (codon 28), and one is in CDR3 (codon 93). This suggests that these substitutions are not critical in determining anti-M specificity. In addition, the library-derived light chains are quite homologous to each other. The amino acid sequence of clone A4 differs from both AP1 and DB1 only at codon 83. There is also a silent mutation at codon 91 that distinguishes clone AP1 from both A4 and DB1.



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  		FIGURE 1. Nucleotide and deduced amino acid sequences of the 425/2B light chain and of the light chains from glycophorin A-specific Fab isolated from the 425/2B Fd:light chain libraries. The A4, AP1, and DB1 light chain sequences are from clones isolated from the 425/2B Fd:light chain libraries. The GenBank accession numbers of A4, AP1, and DB1 are AF005348, AF005349, and AF005350, respectively. The H37-90 light chain is from a highly homologous influenza virus hemagglutinin-specific mAb (41). The upper and lower case letters used to denote the nucleotide sequence indicate replacement and silent codon changes, respectively, from the 425/2B parental light chain sequence.

 
Immunologic characteristics. To investigate the immunologic characteristics of clones isolated from hybridoma Fd:splenocyte light chain libraries, bacterial clones that reacted with M- or N-type glycophorin A in colony-screening assays were expanded, and monoclonal Fab phage were produced and purified. After determining the titers of the different Fab phage preparations, they were each diluted to a stock titer of 1 x 1010 pfu/ml and evaluated by ELISA, using M- and N-type glycophorin A as the target Ags.

To evaluate whether the numbers of Fab molecules on the surface of the Fab phage were similar for the different clones, control ELISA experiments were performed using microtiter plates coated with anti-mouse IgG (for example, see Fig. 2Go). In every case, the different Fab phage preparations revealed similar binding curves. These results suggest that the number and distribution of Fab molecules on the surface of the Fab phage are similar for the different Fab phage preparations tested.



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  		FIGURE 2. Equal titers of Fab phage bind similarly to microtiter plates when analyzed by ELISA. Microtiter plates were coated with anti-mouse IgG as described in Materials and Methods. In A, equal titers of the 425/2B parental Fab phage (circles) and of the A4 Fab phage (squares) isolated from the 425/2B Fd:light chain library were added to corresponding wells. The bound Fab phage were then detected using a combination of biotinylated anti-M13 Ab, peroxidase-conjugated streptavidin, and o-phenylenediamine. The results from analogous experiments comparing the N61 parental Fab phage (circles) and the A1 Fab phage isolated (squares) from the N61 Fd:light chain library are shown in B.

 
An example of the ELISA results examining the anti-M and -N specificity of the Fab phage derived from the 425/2B Fd:anti-M light chain library clones is shown in Figure 3Go. The new A4 Fab phage showed similar anti-M binding specificity and avidity compared with the Fab phage derived from the parental 425/2B hybridoma Ab. This similarity is not surprising given the high degree of homology of the corresponding light chains (Fig. 1Go). Similar results were also obtained with the AP1 and DB1 Fab phage (data not shown).



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  		FIGURE 3. Comparison of binding to glycophorin A of parental, hybridoma-derived Fab phage and of Fab phage derived from the Fd:light chain libraries. Microtiter plates were coated with M-type glycophorin A (open symbols) or N-type glycophorin A (closed symbols) as described in Materials and Methods. In A, equal titers of the 425/2B parental Fab phage (circles) and of the A4 Fab phage (squares) isolated from the 425/2B Fd:light chain library were added to corresponding wells. The bound Fab phage were then detected as described in Figure 2Go. The results from analogous experiments comparing the N61 parental Fab phage (circles) and the A1 Fab phage (squares) isolated from the N61 Fd:light chain library are shown in B.

 
To confirm these results with Fab phage, the relative affinities of the soluble Fab were also examined. To this end, the soluble Fab of 425/2B and AP1 as well as the intact 425/2B mAb were purified. The Fab and the intact IgG were pure when analyzed by SDS-PAGE (Fig. 4GoA, lanes 12,14, and 15); under reducing conditions the light chain and the Fd of each Fab appeared as two distinct bands (Fig. 4GoB, lanes 12, 14, and 15). When tested by ELISA, the AP1 and 425/2B Fab had similar relative affinities for M-type glycophorin A (Fig. 5Go). In addition, both had a 200 to 300 times lower avidity for this Ag compared with the purified 425/2B mAb. The difference in binding between the Fab and the intact IgG mAb (Fig. 5Go) is probably due to the difference in valency between the bivalent IgG mAb and the univalent Fab.



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  		FIGURE 4. SDS-PAGE analysis of purified mAbs and soluble Fab. Hybridoma-derived mAbs, soluble Fab, and Fd:light chain library-derived soluble Fab were purified as described in Materials and Methods. Each of the purified proteins (4 µg) was loaded onto 10% polyacrylamide gels in the absence (A) or the presence (B) of 2-ME, separated by electrophoresis, and visualized with Coomassie blue. The migration positions of the intact mAbs (Mab), intact soluble Fab (Fab), heavy chains (HC), light chains (LC), and Fd (Fd) are indicated. Lane 1, Molecular mass standards (phosphorylase B, 140 kDa; BSA, 67 kDa; OVA, 48 kDa; carbonic anhydrase, 33 kDa; soybean trypsin inhibitor, 29 kDa; lysozyme, 21 kDa); lanes 2 and 11, N92 mAb; lane 3, N92 Fab; lanes 4 and 13, NNA7 Fab; lane 5, C1 Fab; lane 6, G11 Fab; lane 7, N61 mAb; lane 8, N61 Fab; lane 9, A1 Fab; lane 10, 4/3/17, an IgG mouse mAb specific for carcinoembryonic Ag (as an Ig molecular mass standard); lane 12, 425/2B mAb; lane 14, 425/2B Fab; lane 15, AP1 Fab.

 


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  		FIGURE 5. Comparison of binding to glycophorin A of parental, hydridoma-derived mAbs, of parental, hydridoma-derived soluble Fab, and of soluble Fab derived from the Fd:light chain libraries. Microtiter plates were coated with M-type glycophorin A (open symbols) or N-type glycophorin A (closed symbols), and mAbs and soluble Fab were purified as described in Materials and Methods. In A, defined concentrations of the 425/2B parental mAb (squares), the 425/2B parental-derived soluble Fab (circles), and the AP1-soluble Fab (triangles) isolated from the 425/2B Fd:light chain library were added to corresponding wells. The bound Ig proteins were then detected using peroxidase-conjugated anti-mouse Fab followed by o-phenylenediamine. The results from analogous experiments comparing the N61 parental mAb (squares), the N61 parental-derived soluble Fab (circles), and the A1 soluble Fab (triangles) isolated from the N61 Fd:light chain library are shown in B.

 
Chimeras isolated from the N61 Fd:anti-N light chain libraries

Sequence information. When the N1 and N3 light chain libraries, obtained from the spleens of two mice immunized with N-type glycophorin A, were each coupled with the Fd of the N61 mAb and then panned on N-type glycophorin A, two types of clones reacting with N-type glycophorin A were found. One type contained a light chain with a sequence identical with that of the N61 hybridoma Ab (24). We assume that these clones resulted from slight contamination of the light chain libraries with the original N61 light chain due to incomplete digestion of the parental plasmid with restriction endonucleases; the contaminant was then enriched and selected for by the panning method. The second type, represented by clone A1, contains a light chain that is a product of the Vk10 gene family and is thus similar to the N61 parental light chain. However, A1 uses the Jk5 gene segment instead of Jk4 found in the N61 light chain (Fig. 6Go). The homology in amino acid sequence between the V regions of the N61 and A1 light chains is 97%. Although the A1 and N61 light chains are identical with each other at codons 1 through 8, the amino acid sequence differences between them and the germ-line sequence at these positions are attributable to the oligonucleotide primers used for amplification. The A1 light chain differs from the N61 light chain by five nucleotide substitutions at codons 30, 34, 44, 49, and 53 (Fig. 6Go). Interestingly, at these codons the A1 sequence is identical with the known germ-line sequence. Since Fab containing the N61 Fd and either the N61 or A1 light chains both recognize N-type glycophorin A, the four amino acid changes at codons 30, 34, 44, and 49 are probably not required for anti-N specificity. However, only two of these differences, at codons 30 and 34, occur in a complementarity-determining region (i.e., CDR1).



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  		FIGURE 6. Nucleotide and deduced amino acid sequences of the N61 light chain and the light chain from a glycophorin A-specific Fab isolated from the N61 Fd:light chain libraries. The A1 light chain sequence is from a clone isolated from the N61 Fd:light chain libraries. The GenBank accession number of A1 is AF005354. The Vk10 germ-line sequence Vk10-Ars-A is also shown (76).

 
After panning both of the two N61 Fd:anti-N light chain libraries on M-type glycophorin A, no clones reacting with either M- or N-type glycophorin A were found.

Immunologic characteristics. The ELISA results examining the anti-M and -N specificity of the N61-derived Fab phage demonstrated that the two had similar anti-N specificities, whereas the A1 Fab phage appeared to have less avidity than the N61 Fab phage for M-type glycophorin A (Fig. 3Go). To examine the relative affinities of the N61 and A1 Fab further, soluble Fab as well as intact IgG N61 mAb were purified (Fig. 4Go, lanes 7–9). When tested by ELISA, the purified N61 and A1 Fab were similar to each other in their affinities for N-type glycophorin A (Fig. 5Go). In addition, both had a 200 to 300 times lower avidity for M- and N-type glycophorin A compared with the purified mAb (Fig. 5Go). The difference in binding between the Fab and the mAb is probably due to the difference in valency between the mAb and the Fab. Thus, these results are quite similar to those obtained above with the 425/2B-derived Fab.

Sequence information. The light chain libraries from each of two different mice immunized with N-type glycophorin A were linked with the Fd of the N92 mAb and then panned on N-type glycophorin A. Three different types of clones reacting with N-type glycophorin A were obtained, represented by NNA7, C1, and G11 (Fig. 7Go). Clones NNA7 and G11 were obtained using the N1 light chain library; C1 was derived from the N3 library (Table IIIGo). As was found with the N92 hybridoma Ab, the light chains from each of the three new clones were encoded by a Vk1 germ-line gene. In addition, the NNA7, C1, and G11 light chains used the Jk1 or Jk5 gene segments, in contrast to the original N92 light chain, which used the Jk4 gene segment. The complete nucleotide sequences of these light chains compared with those of two published germ-line sequences (K5.1 and K1A5) are shown in Figure 7Go. The amino acid sequence differences at codons 1 through 7 between the new clones and either the N92 or the germ-line sequences are attributable to the sequences of the different oligonucleotide primers used for amplification. It is noteworthy that the N92 light chain is two amino acid residues shorter than the NNA7, C1, and G11 light chains; this may have been caused by differential annealing of the primer used for amplification. The new clones demonstrate amino acid sequence homology with the V region of the N92 light chain of 92% for C1 and 95% for each of NNA7 and G11. Most of the nucleotide differences between the N92 light chain and NNA7, C1, and G11 occur at positions where the N92 light chain differs from at least one of the germ-line sequences (e.g., codons 28, 40, 52, 74, 87, 91, and 92). Four of the N92 light chain replacement mutations were in complementarity-determining regions: one in CDR1 (i.e., at codon 28) and three in CDR3 (i.e., at codons 89, 91, and 92). However, with regard to CDR3, an argument could be made that the N92 light chain may be derived from the K5.1 germ-line gene in the Vk1-A subgroup and that NNA7, C1, and G11 may be derived from the K1A5 germ-line gene in the Vk1-C subgroup (Fig. 7Go). In addition, outside of codons 1 through 7, NNA7 and G11 have an identical amino acid sequence, and the amino acid sequence of C1 differs from those of both NNA7 and G11 at only four codons: 28, 96, 100, and 106 (Fig. 7Go).



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  		FIGURE 7. Nucleotide and deduced amino acid sequences of the N92 light chain and the light chains from glycophorin A-specific Fab isolated from the N92 Fd:light chain libraries. The NNA7, C1, and G11 light chain sequences are from clones isolated from the N92 Fd:light chain libraries. The GenBank accession numbers of NNA7, C1, and G11 are AF005351, AF005352, and AF005353, respectively. The two most homologous Vk1 germ-line sequences, K5.1 and K1A5, are also shown (77). The third germ-line gene sequence in this family, K18.1, is less homologous (77) and is not shown. The K5.1 and K1A5 sequences have also been classified as belonging to the Vk1-A and Vk1-C subgroups, respectively (78).

 
Finally, as with the N61 Fd:anti-N light chain libraries, no glycophorin A-reactive clones were found after panning of the N92 Fd:anti-N light chain libraries on M-type glycophorin A.

Immunologic characteristics. The ELISA results examining the anti-M and -N specificity of the N92-derived Fab phage are shown in Figure 8Go. The N92, C1, NNA7, and G11 Fab phage all showed anti-N specificity. However, the Fab phage obtained from the N92 Fd:anti-N light chain libraries (C1, NNA7, and G11) showed higher avidity for N-type (and M-type) glycophorin A than did the parental N92 Fab phage (Fig. 8Go, B and C). This result was obtained even though the Fab phage preparations from the different clones were normalized by ELISA (Fig. 8GoA) and titer.



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  		FIGURE 8. Comparison of binding to glycophorin A of parental, hydridoma-derived N92 Fab phage and of Fab phage derived from the N92 Fd:light chain libraries. To control for phage titer, microtiter plates were coated with anti-mouse IgG (A). To measure binding of Fab phage to glycophorin A, microtiter plates were coated with N-type (B) or M-type (C) glycophorin A as described in Materials and Methods. In each panel, equal titers of the N92 parental Fab phage (squares) and the C1 (closed circles), NNA7 (open circles), and G11 (triangles) Fab phage isolated from the N92 Fd:light chain libraries were added to corresponding wells. The bound Fab phage were then detected as described in Figure 2Go.

 
To examine whether the increased binding of the library-derived Fab phage to glycophorin A was due to changes in avidity or affinity, soluble Fab were prepared. As was found with the 425/2B-derived Fab, the N92 light chain and the Fd appeared as two distinct bands when examined by SDS-PAGE in the presence of 2-ME (Fig. 4GoB,lane 3); similar results were found with NNA7, C1, and G11 (Fig. 4GoB, lanes 4–6). As expected, when tested by ELISA, the N92 Fab had a 100 to 200 times lower avidity for N-type glycophorin A than the parental N92 IgG mAb; no binding to M-type glycophorin A was seen (data not shown). In contrast, the library-derived Fab (NNA7, C1, and G11) bound to N-type glycophorin A with relative affinities significantly higher than that of the N92 Fab (Fig. 9GoA) and similar to that of the N92 mAb (Fig. 9GoB). These results were reproducible even when different preparations of Fab were prepared from the same clone (data not shown). As with the Fab phage (Fig. 8Go), the C1, NNA7, and G11 Fab exhibited slight binding to M-type glycophorin A (data not shown). Interestingly, the increase in affinity found with the library-derived clones allowed these univalent Fab to bind to N-type glycophorin A as efficiently as the bivalent N92 parental hybridoma mAb (Fig. 9GoB). This increase in affinity was also manifested by the ability of these Abs to function in a Western blotting assay (Fig. 10Go). Thus, although the N92 mAb (at 100 nM) was able to detect human RBC glycophorin A (and glycophorin B) on blots (Fig. 10Go, lanes 1 and 2), the N92 Fab was ineffective in this assay even at a concentration of 200 nM (Fig. 10Go, lanes 3 and 4). In contrast, the NNA7 Fab was a very sensitive probe in this assay, yielding a strong signal, even at a concentration as low as 10 nM (Fig. 10Go, lanes 5 and 6). Taken together, these results suggest that the somatic mutations found in the N92 light chain compared with those in the germ-line configuration may have led to a decreased affinity of the N92 Fab for N-type glycophorin A. Alternatively, the use of the Jk1 or Jk5 gene segments may confer a higher affinity for N-type glycophorin A than that found when the Jk4 gene segment is coupled with the K5.1 or K18.1 germ-line genes.



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  		FIGURE 9. Comparison of binding to glycophorin A of the parental N92 mAb, parental N92-derived soluble Fab, and soluble Fab derived from the N92 Fd:light chain libraries. mAbs and soluble Fab were purified, and microtiter plates were coated with N-type glycophorin A as described in Materials and Methods. Defined concentrations of the N92 parental mAb (closed circles), the N92 parental-derived soluble Fab (open squares), and the G11 (open circles), NNA7 (triangles) and C1 (closed squares) soluble Fab isolated from the N92 Fd:light chain libraries were added to corresponding wells. The bound Ig proteins were then detected as described in Figure 5Go. In A, all the soluble Fab are compared with each other; in B, the library-derived Fab are compared with the parental, hybridoma-derived mAb. The experiments in A and B were performed on separate occasions.

 


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  		FIGURE 10. Detection of glycophorin A on Western blots using N92 mAb, N92 Fab, and NNA7 Fab. M-type glycophorin A (lanes 1,3, and 5) and N-type glycophorin A (lanes 2, 4, and 6) were purified from human RBCs, separated by SDS-PAGE, and blotted onto nitrocellulose paper. The blots were then probed with 100 nM N92 mAb (lanes 1 and 2), 200 nM N92 Fab (lanes 3 and 4), and 10 nM NNA7 Fab (lanes 5 and 6). The migration positions of the glycophorin A monomer and dimer and of the glycophorin B monomer and dimer are shown. Glycophorin B, which is also present in small amounts in these preparations, encodes the N blood group Ag even when it is isolated from individuals who are homozygous for the M blood group Ag; this explains the reactivity in lanes 1 and 5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There has been increasing interest in understanding the immune response to glycopeptide epitopes (4, 5, 6, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52). The human MN Ags are clinically relevant glycopeptide Ags involved in hemolytic transfusion reactions and hemolytic disease of the newborn (53, 54). Studies of Ab fine specificity demonstrated that binding of many Abs to the MN Ags is exquisitely dependent on the structure of these Ags (14, 17, 18, 19, 21, 22, 23). For example, the binding of N61 depends on both a leucine at position 1 and sialic acids on the O-glycans attached to the amino acids at positions 2 to 4 of glycophorin A (21, 22).

To begin studying the immune response to the MN Ags, we previously sequenced eight murine mAbs obtained from mice immunized by different protocols (24). Because all four anti-N mAbs used the same JH gene segment and heavy chain V regions derived from the same VH gene family, these results suggested that the immune response to the N Ag was restricted. Since these four anti-N mAbs used light chain V regions derived from three different Vk gene families, this suggested that N-specific heavy chains might promiscuously pair with various light chains to yield Ag specificity. This hypothesis was tested in a limited way using Fab phage methodology (25). Although these light chain shuffling studies suggested the importance of particular heavy chain:light chain pairing for providing anti-N specificity, the results of this limited investigation were not conclusive (25). Therefore, in the current report, particular hybridoma-derived anti-N and anti-M Fd were deliberately coupled with light chains derived from mice immunized with glycophorin A. This provides a robust test of the degree of flexibility in heavy chain:light chain pairing that can yield MN specificity. In addition, using light chains from immunized mice may increase the chance of finding heavy chain:light chain pairs with the desired specificity (32, 39). These light chain shuffling experiments demonstrated that for an Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the corresponding, parental, hybridoma-derived mAb (Figs. 1Go, 6Go, and 7Go). In addition, the homology in amino acid sequence between the parental light chain and the library-derived light chains was 92 to 97%. The overall MN Ag specificity of the library-derived Fab phage was the same as that of the parental mAb (Table IIIGo and Figs. 3Go, 5Go, 8Go, and 9Go). Nonetheless, in certain cases the new light chains did modify the Fab affinity and fine specificity (for example, see Fig. 9Go). Taken together, this demonstrates that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain to create a functional binding site for these glycopeptide Ags. Thus, these new results demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and -N Abs.

Determining the relative contributions of heavy and light chains in forming an Ab with particular Ag specificity is a classical problem in immunology. It was originally studied using methods for separating and recombining light chains and heavy chains from polyclonal sera (39, 55) and myeloma proteins (39, 56). These early studies, which primarily evaluated Abs directed against a hapten, DNP, yielded conflicting results, suggesting the presence (39) and absence (56) of heavy chain promiscuity. Recent studies used recombinant DNA approaches to analyze the role of heavy chain promiscuity in the binding of mouse and human mAbs or Fab to haptens (32, 57, 58) and to peptide (1, 3, 59, 60, 61, 62, 63), nucleic acid (2, 64, 65, 66), and carbohydrate or glycopeptide (25, 67) Ags. As extreme examples of the potential dominance of the heavy chain in determining Ag specificity, in some cases the heavy chain V region alone can bind Ag (64, 68). This is supported by multiple studies demonstrating that a given heavy chain can combine promiscuously with multiple light chains and yet retain Ag specificity (1, 32, 57, 58, 61, 64, 65, 66). However, in some cases the fine specificity of the chimeric Abs was somewhat different from that of the parental Ab (65, 66). In contrast, multiple studies of Abs against peptide (3, 59, 60, 62), nucleic acid (2), and carbohydrate or glycopeptide (25, 67) Ags showed a lack of heavy chain promiscuity. The most convincing of these coupled a light chain library with a given heavy chain and tested the resulting clones for Ag binding (2, 3, 59, 60). For example, a heavy chain from a human mAb directed against the thyroid peroxidase autoantigen was coupled with a light chain library derived from thyroid-infiltrating lymphocytes isolated from a patient with Graves’ disease (3, 60). All 11 resulting thyroid peroxidase-binding clones used a light chain from the same Vk1 germ-line gene family as the parental mAb, and the light chains from two clones were identical with the parental light chain. In addition, the sequences of some of the library-derived light chains had a more germ-line-like configuration than the parental light chain (3); this is similar to some of our results (Fig. 7Go). Other studies showed that even when there is a lack of heavy chain promiscuity, and the new light chains are highly homologous to the parental light chain, the resulting mAbs or Fab may have a somewhat lower affinity or altered fine specificity, even while retaining identical overall Ag specificity (2, 59, 66). Again, these results are similar to those described in the current report.

A general explanation is not yet available as to why a given heavy chain can or cannot promiscuously pair with various light chains to yield an Ag-specific Ab. The chemical nature of the Ag may or may not be important. For example, heavy chain promiscuity was found for Abs directed against protein Ags (1) and nucleic acids (66); however, the opposite was also found for both protein Ags (3) and nucleic acids (2). Nonetheless, since the chemical nature of the Ag may direct the details of its interaction with Ab, this may yet explain the existence of heavy chain promiscuity. As first suggested by Marcus (67) for carbohydrate Ags, heavy chain promiscuity may depend on the number of contacts between Ag and Ab and the extent of the Ab surface area that is buried after interaction with Ag. Since anti-carbohydrate mAbs make few contacts with Ag (69, 70, 71, 72, 73), this not only explains the low affinity of anti-carbohydrate Abs, but also suggests that if any contacts are disrupted by light chain shuffling, Ag specificity will be lost. Whether this also applies to glycopeptide Ags must await a better understanding of the molecular interactions between Abs and these Ags.

The selection of a functional light chain by a given anti-M or -N Fd was not random, suggesting that the conformation of the Ab binding site requires specific light chain structures. Interestingly, for the N92 mAb, most of the amino acid differences between the library-derived light chains and the hybridoma-derived light chain occurred where the sequence of the latter differed from the published germ-line sequences (Fig. 7Go). That is, the library-derived light chains had a more germ-line-like configuration (3). In addition, when comparing the CDR amino acid sequences between the library-derived and the hybridoma-derived light chains, the differences occurred in CDR1 and CDR3, but not in CDR2. This agrees with previous studies (74), which found that most light chain somatic mutations that arise during affinity maturation occur in CDR1 and CDR3. Finally, since most of the differences between germ-line gene sequences and the sequences of mature, rearranged light chains are caused by somatic mutations that arise during affinity maturation (74, 75), it may be that some mutations in the N92 light chain (Fig. 7Go) do not play an important role in increasing the affinity of this anti-N Ab (Fig. 9Go). Indeed, some of these mutations may actually lead to a decreased affinity for Ag.

Previous studies suggested that chain shuffling may be useful for obtaining Fab (or mAbs or Fab phage) that have increased affinity and yet retain Ag specificity (32, 59, 61). In our case, shuffling of the N92 light chain resulted in library-derived Fab with increased affinity (Fig. 9Go). In addition, the affinities of these univalent library-derived Fab were equivalent to that of the parental, bivalent, N92 hybridoma IgG Ab (Fig. 9Go). This approach may have clinical and practical applications. For example, the NNA7 Fab was at least as effective as the N92 mAb in Western blotting (Fig. 10Go). In addition, the NNA7, G1, and C11 Fab were as effective as the N92 mAb in typing of human MM, MN, and NN RBC by hemagglutination methods; in contrast, the N92 Fab did not agglutinate any human RBC (M. Czerwinski, D. L. Siegel, and S. L. Spitalnik, manuscript in preparation). This suggests that soluble Fab of sufficient affinity, which are produced by bacterial culture, may be able to substitute in clinically relevant assays for monoclonal or polyclonal reagents that are currently obtained using more expensive methods, such as mammalian cell culture.


   Acknowledgments
 
We thank Dr. Jonni S. Moore for help with mouse immunization and splenocyte isolation, Dr. Fritz Grunert for the gift of Ab 4/3/17, and Dr. Elwira Lisowska for the gift of purified glycophorin A and the N61, N92, and 425/2B hybridomas.


   Footnotes
 
1 This work was supported in part by grants from the Polish Committee of Scientific Studies (Konfederacja Polski Niepodleglej Grant 6P04A 05608) and from the National Institutes of Health (R01HL46206 and P50HL54516). Back

2 Address correspondence and reprint requests to Dr. Steven L. Spitalnik, Department of Pathology and Laboratory Medicine, 220 John Morgan Building, University of Pennsylvania, Philadelphia, PA 19104. E-mail address: Back

3 Abbreviations used in this paper: TBS, Tris-buffered saline; pfu, plaque-forming units. Back

Received for publication August 8, 1997. Accepted for publication January 5, 1998.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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