The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, Y. W.
Right arrow Articles by Margolies, M. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, Y. W.
Right arrow Articles by Margolies, M. N.
The Journal of Immunology, 1998, 160: 5990-5997.
Copyright © 1998 by The American Association of Immunologists

Structural Requirements for a Specificity Switch and for Maintenance of Affinity Using Mutational Analysis of a Phage-Displayed Anti-Arsonate Antibody of Fab Heavy Chain First Complementarity-Determining Region1

Yee Wah Wong*, Davinder S. Gill{dagger}, Behnaz Parhami-Seren{ddagger}, Mary K. Short§, Seshi Reddy Sompuram and Michael N. Margolies2,{ddagger}

* Department of Obstetrics and Gynecology, The Ohio State University, Columbus, OH 43120; {dagger} IntraImmune Therapies, Boston, MA 02215; {ddagger} Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; § Department of Hematology/Oncology, Children’s Hospital Medical Center, Cincinnati, OH 45229; and Hubert Humphrey Cancer Center and Department of Pathology, Boston University School of Medicine, Boston, MA 02118


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30–36 in Ab 36–65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36–65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36–71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline VH sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
When A/J mice are immunized with p-azophenylarsonate (Ars)3 protein conjugates, the majority of the serum Abs in secondary responses express a heritable cross-reactive Id (1), designated IdCR. These Abs are each encoded by a single combination ("canonical") of germline V region gene segments (2, 3, 4). These canonical Abs, which differ from each other only in somatic mutations and at heavy (H) chain V region gene junctions, have proved useful models for the analysis of Ag-driven somatic mutation resulting in affinity maturation (2, 5, 6, 7). Using site-directed mutagenesis, it has been possible to increase affinity for Ars in vitro through amino acid replacements that are not observed in vivo following immunization (8) and to change fine specificity (9). Immunization with p-azophenylsulfonate (Sulf)-protein conjugates does not result in the expression of Sulf-binding canonical Abs, unless there was prior immunization with Ars (10). Wysocki and coworkers (11) showed that immunization with Ars-protein conjugates, followed by immunization with Sulf-protein conjugates, resulted in the isolation of three mAbs that bound Sulf, but not Ars, yet used the canonical gene combination typically associated with Ars-binding Abs. The sequences of these three mAbs contained a recurrent somatic mutation at position H:35 that formed the basis for site-directed mutagenesis of canonical Abs to engineer a switch in fine specificity from Ars to Sulf (9). In both the unmutated Ab 36–65 (5) and the somatically mutated higher affinity anti-Ars IdCR+ Ab 36–71, for which the Fab x-ray crystal structure was determined (12, 13), the single mutation of H:Asn35, a putative contact residue in the first heavy chain complementarity-determining region (HCDR1), to Ala caused a change in specificity from Ars to Sulf (9). To further probe the contribution of HCDR1 to Sulf specificity and to determine the degree of sequence stringency consonant with the specificity change, we generated a panel of 36–65 Fab with random HCDR1 sequences displayed on filamentous bacteriophage (14, 15). Sulf-specific Fab were isolated by panning, their sequences were determined, and Ag binding was characterized. Based on these results, site-directed mutants were generated to examine the role of residues H:35 in specificity and H:33 in affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The production of the murine A/J hybridoma cell line 36–65 (IgG1,k) and its H chain loss variant (36–65L) was previously described (5, 16). The hybridoma cell line HP Sulf-1 (11) was a gift of L. Wysocki (National Jewish Center for Immunology, Denver, CO). The Sulf-binding transfectoma cell line 36–65 containing the H chain mutations V100H/Y107S/N35A4 was described (9, 17). An Ars-binding variant of 36–65 containing the H chain junctional mutations V100H and Y107S, as well as the Sulf-binding mutants derived therefrom containing H:N35A, was described (9). The 36–65 transfectoma cell line with a polyglycine-replaced HCDR1, as well as the mutant derived therefrom containing the back mutation G35N, has also been described (18).

Synthesis and purification of haptens and hapten conjugates

p-aminophenylarsonate (Ars), p-aminophenylphosphonate (Phos), and p-aminophenylsulfonate (Sulf) were obtained from Aldrich (Milwaukee, WI). The N-acetyl-L-tyrosine derivatives of Ars, Sulf, and Phos were prepared as described (19, 20). Hapten conjugates of bovine {gamma}-globulin (BGG) were prepared as described (21).

Construction of library of HCDR1 mutants in bacteriophage

Total mRNA was extracted from 36–65 hybridoma cells, and the cDNA of the Fd (VH+CH1) and light (VL+CL) chain of Ab 36–65 was PCR amplified using the following primers: for the H chain, H1, 5'-ATGGCCGAGGTTCAATTGCAGCAGTCTGGAGC-3'; and H2, 5'-CTGTACATATACTAGTCTTAGAACCACAATCC-3'. For the L chain, L1, 5'-CAACCAGCCATGGCCGATATCCAGATGACACAGACTACATCCTCC-3'; and L2, 5'-GGCTAGTCTAGATCATTAACACTCATTCCTGTTGAAGCTCTTGACGATGGG-3' (restriction sites are underlined). The PCR fragments of H and L chains were digested with Mun I/SpeI and NcoI/XbaI, respectively. The pComb3 vector (Fig. 1Go, a gift of Dr. Carlos Barbas, The Scripps Research Institute, La Jolla, CA) (22) was modified by adding a MunI site and deleting an upstream NcoI site without changing the amino acid sequence. The digested light chain DNA was first ligated into the NcoI/XbaI-digested vector. After confirming the sequence, the H chain was ligated into the MunI/SpeI-digested vector containing the L chain. Primers H1 and L1 contain codons at the H and L chain N termini designed to reproduce the correct 36–65 N-terminal sequences (Fig. 1Go). To randomly mutagenize the CDR1 of the 36–65 H chain (HCDR1), two restriction enzyme sites, HindIII and XhoI, were constructed, flanking the HCDR1 sequence using two primers: R1, 5'-ATGTCCTGCAAAGCTTCTGGATA-3'; and R2, 5'-ACAGGGCCTCGAGTGGATTGGATA3'. The seven amino acid random sequences of the HCDR1 region were synthesized by PCR using these two primers with the oligonucleotide 5'-AGCTTCTGGATATACATTC(NNS)7GTGAAACAGAGGCCTGGACAGGGCC-3' as template, in which N represents an equimolar ratio of the four dNTPs and S represents an equimolar ratio of dGTP and dCTP. The synthesized fragments were digested with HindIII/XhoI and ligated into the HindIII/XhoI-digested vector.



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Schema of the vector construction and saturation mutagenesis of the HCDR1 of 36–65 Fab. The VH-CH1 segment of the 36–65 H chain was cloned into the pComb3 vector (22) using MunI/SpeI cloning sites. The randomized region of HCDR1 (residues 30–36) was constructed using HindIII/XhoI cloning sites. The L chain was cloned into pComb3 using NcoI/XbaI cloning sites. The positions of the oligonucleotides used in cloning are denoted by arrows (see text for details).

 
The above construct was electroporated into Escherichia coli BB4 cells. After growth at 37°C for 1 h, the culture was supplemented with 100 µg/ml carbenicillin (Carb) and 10 µg/ml tetracycline (Tet) and grown for another hour. The culture was then infected by helper phage VCSM13 (Stratagene, La Jolla, CA), and a fraction of the culture was plated onto LB/ampicillin agar plates to determine the titer of the library, while the rest of the culture was shaken for 2 h before adding 70 µg/ml kanamycin and incubated overnight. The supernatant was harvested by centrifugation at 7000 rpm for 5 min at 4°C. Phage were harvested by adding 4% (w/v) polyethylene glycol 8000/0.8 M sodium acetate, pH 7.5, at 4°C for 30 min and then centrifuged. The pellet was resuspended in TBS/1% BSA and stored at -20°C.

Selection of Sulf-specific mutant Abs from phage libraries

Sulf-BGG was coupled to Sephacryl-300, as described (23). A total of 1 ml of the resultant affinity resin was incubated with 2 ml of 3% BSA/TBS for 1 h at room temperature with head-to-tail mixing. The resin was then mixed with 1 ml phage and incubated for 2 h. The resin was transferred into a column and washed with 60 ml 0.5% Tween-20/TBS. The bound phage were eluted with 10 ml 100 mM aqueous solution of triethylamine and immediately neutralized with 5 ml 1 M Tris, pH 7.5. The collected eluent was mixed with 15 ml of E. coli BB4 (OD600 = 0.6–0.8) for 15 min at room temperature. An aliquot was plated onto Carb/Tet/LB plates, and the remaining culture was incubated at 37°C for 1 h with 20 µg/ml Carb and 10 µg/ml Tet. The Carb concentration was adjusted to 100 µg/ml, and incubation was continued for an additional hour. One milliliter of VCSM13 helper phage (1012 plaque-forming units/ml) was then added; the culture was incubated for 1 h before adding 70 µg/ml kanamycin, and then left overnight. The phage were harvested as described above, and the selection procedure was repeated three times with fresh Sulf-BGG resin to enrich Sulf-specific binding clones.

Screening of selected Sulf-binding clones by ELISA

After four rounds of selection, clones were picked from the LB plates and grown in 2 ml Carb/Tet/superbroth for 3 h at 37°C. Ten microliters of VCSM13 helper phage were added and incubated for another 2 h before adding kanamycin at a final concentration of 70 µg/ml. The culture was then grown overnight. Phage supernatants were harvested by centrifugation, as described above. PVC plates were coated with 100 µl/well of 2 µg/ml Sulf-BGG or goat anti-mouse IgG (G{alpha}M) F(ab')2 (ICN, Costa Mesa, CA) and incubated overnight at 4°C. The wells were rinsed three times with 200 µl 0.05% Tween-20/PBSA (0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.02% NaN3, pH 7.4) and then blocked with 150 µl 3% BSA/0.05% Tween-20/PBSA and incubated at 37°C for 1 h. The blocking solution was replaced with 100 µl phage supernatant and incubated at 37°C for 2 h. The wells were washed three times with 200 µl 0.05% Tween-20/PBSA, and 100 µl of horseradish peroxidase (HRP)-conjugated goat anti-M13 Abs (Pharmacia, Piscataway, NJ) was added and incubated for 1 h at 37°C. The wells were then washed three times with 200 µl 0.05% Tween-20/PBSA; 100 µl of TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and the reaction was terminated by adding 50 µl 2 M H3PO4. Color development was determined at 450 nm using a Bio-Tek ELISA reader (Bio-Tek Instruments, Winooski, VT). Clones were selected for further study if the OD was >=1 for Sulf-BGG and G{alpha}M F(ab')2. All positive clones selected bound equally well to both Sulf-BGG and G{alpha}M F(ab')2, while the negative clones were all at background levels (OD <0.002). To determine the specificity of the selected Sulf-binding clones, an ELISA was performed on phage-displayed Fab with plates coated with 100 µl/well of 2 µg/ml Ars-BGG, Sulf-BGG, Phos-BGG, G{alpha}M F(ab')2, and BGG (as negative control).

Expression and purification of soluble Sulf-binding Fab fragments

The plasmids of selected Sulf-binding clones were digested with SpeI/NheI to excise the gene III DNA fragment (Fig. 1Go). The plasmid was self ligated and electroporated into E. coli BB4 cells. Each selected clone was grown in 1 L Carb/Tet/superbroth at 37°C overnight. The cells and supernatant were separated by centrifugation. The cells were resuspended in 100 ml of lysis buffer (50 mM glucose, 0.5 mg/ml Pefabloc, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin (Boehringer Mannheim, Indianapolis, IN), 25 mM EDTA, and 25 mM Tris-HCl, pH 8) and incubated with 2.5 mg/ml lysozyme for 15 min on ice. Brij 58 was added to a concentration of 0.5%, and the mixture was left on ice for 30 min. The supernatant was harvested by centrifugation at 16,000 rpm for 30 min at 4°C. The Sulf-binding Fab was purified from the supernatant by affinity chromatography with Sulf-BGG-conjugated Sepharose, as described (9). The purification of the 36–65 mutant with polyglycine-replaced HCDR1 (36–65 H1Gly) and the back-mutant H1Gly/G35N has been described (18). The mutants 36–65 H1Gly/G35A, 36–65 V100H/Y107S/N35A/G33A, and S3 G33A (see Table IIIGo) were purified using Sulf-BGG-conjugated Sepharose, as described (9). The purity and concentration of the IgGs and Fab were determined by SDS-PAGE gel electrophoresis under reducing and nonreducing conditions, and absorbance at 280 nm.


View this table:
[in this window]
[in a new window]
  		Table III. Binding affinities of HCDR1 site-directed mutants1

 
Determination of Ab affinity and avidity

Binding affinity (Ka) of Abs and Fab fragments for Ars-N-acetyl-L-tyrosine and Sulf-N-acetyl-L-tyrosine was determined by fluorescence quenching, as reported (24, 25). Ka values reported are averages of at least three determinations. The Fab obtained by enzymatic digestion from the mAbs 36–65 and HP Sulf-1 were used as controls. The direct binding of Abs and Fab fragments to Ars-BGG and Sulf-BGG was determined in triplicate by ELISA. The 96-well PVC plates were coated with 100 µl of 2 µg/ml Ars-BGG or Sulf-BGG, and 100 µl of serially diluted purified Abs or Fab fragments (0.64–50,000 ng/ml; fivefold dilutions) was added. Binding was detected by adding HRP-conjugated goat anti-mouse F(ab')2 (Jackson ImmunoResearch, West Grove, PA) and stopped by addition of 50 µl 2 M H3PO4. The end point of the reaction was determined in an ELISA reader at 450 nm. The concentration of each Fab fragment at which 50% binding was achieved was used in a competition ELISA assay.

The relative affinity was measured in triplicate by competition ELISA. The 96-well PVC plates were coated with 100 µl of 2 µg/ml Ars-BGG or Sulf-BGG. A total of 50 µl of Fab (concentrations as determined above) was added to each well with 50 µl of serially diluted Ars-N-acetyl-L-tyrosine or Sulf-N-acetyl-L-tyrosine (twofold dilutions, 0.8–500 nM) and incubated at 37°C for 2 h. The binding of Fab to the plate was determined by adding HRP-conjugated G{alpha}M F(ab')2, as described above. The concentration of Ars-N-acetyl-L-tyrosine or Sulf-N-acetyl-L-tyrosine required for 50% inhibition of binding of Fab to the coated plate was determined.

Site-directed mutagenesis of mutant Fab in pComb3

For the H:G33A site-directed mutation of both the anti-Ars 36–65 Fab and a Sulf-binding mutant Fab designated S3 (Table IIIGo), two oligonucleotides were synthesized with sequences matching a 65-bp region across HCDR1, except for the residue to be mutated. These oligonucleotides were then used as a template in separate PCR reactions using a 26-mer sense primer bearing a HindIII site and a 26-mer antisense primer bearing an XhoI site (Fig. 1Go). The PCR products were gel purified and digested with HindIII/XhoI. The digested products were repurified and ligated into pComb3 containing the appropriate Fab that had been digested with HindIII/XhoI. The vector containing the site-directed mutation was introduced into XLI-Blue E. coli cells. The presence of the mutations was verified by dideoxy chain termination DNA sequencing.

Mutagenesis and expression of mutant Abs in hybridoma cells

As described previously (25, 26, 27), mutations (indicated in parentheses) were introduced into the cloned 36–65 VDJ gene (H:G33A, H:G33L), the 36–65 VDJ gene containing the mutations N35A/V100H/Y107S (9, 17) (H:G33A), or the 36–65 polyglycine HCDR1/G35N (18) (H:G35A). The mutant genes were subcloned into an expression vector (28) and transfected into the L chain-producing hybridoma cell line 36–65 L by electroporation and selected as described (24, 27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of randomized HCDR1 region of 36–65 Fab

The cDNA fragment representing 36–65 Fab was generated by PCR amplification and subcloned into the pComb3 vector (Fig. 1Go). The resulting construct was transfected into E. coli BB4 cells, and phage particles were harvested. The binding activity of the wild-type (wt) Fab fragment displayed on phage was confirmed by direct binding to Ars-coated plates by ELISA (data not shown). The HCDR1 region (amino acids 31–35 plus the flanking framework residues 30 and 36 (29)) of the cloned 36–65 H chain Fd fragment (VH+CH1) was subjected to saturation mutagenesis using PCR. The randomization of this region was confirmed by sequencing pooled DNA from the library. Equal intensity of all four bases was observed for the first and second bases, while the third base for each mutagenized codon showed equal proportions of G and C (corresponding to synthesis of NNS codons; see Materials and Methods; data not shown). The randomized construct was electroporated into bacteria, and phage particles were harvested. Based on transformation efficiency, the nominal size of this randomized HCDR1 library was >=2 x 107 clones.

Selection of Sulf-specific phage clones

One milliliter of phage library (>1012 plaque-forming units/ml) was used in selection using Sulf-BGG-conjugated Sephacryl resin. After the fourth round of selection, the eluted phage were transfected into bacteria, and independent colonies were picked and grown with helper phage. The phage supernatant was harvested, and binding to Sulf was determined by ELISA on Sulf-BGG-coated plates. Ninety-six clones were screened; 55 bound Sulf-BGG. These positive clones were sequenced, revealing seven different DNA sequences. The amino acid sequences and the frequency of appearance are shown in Table IGo. Among clones with identical amino acid sequences, in all cases only a single nucleotide sequence was observed. At position 30, six different amino acid residues were observed. At position 31, five different amino acids occurred with three of seven sequences containing Gly. At position 32, four different amino acids were identified, with aromatic residues found in five of seven mutants. At position 33, glycine occurred in all seven sequences, encoded by two different codons. At position 34, three different amino acids were found. Five of seven mutants contained leucine at position 34, while the other two substitutions were also hydrophobic. At position 35, three different aliphatic residues occurred: isoleucine, alanine, and valine. We had reported previously that single mutations at position 35 to Ser, Thr, Ala, and Cys in Ab 36–65 resulted in a specificity change from Ars to Sulf (9).


View this table:
[in this window]
[in a new window]
  		Table I. Amino acid sequences of the HCDR1 region of Sulf-binding mutants and frequency of their appearance in selected phage library1

 
The fact that identical nucleotides were used to encode the same amino acid sequences of mutants that were selected more than once raises the possibility that the library size was limiting and/or that screening or selection bias occurred. Nonetheless, the data were sufficient to serve as a basis for the design of site-directed mutants to examine the requirements in HCDR1 for Ars and Sulf binding. The high frequency of clone S3 (42/55) may be related to its higher affinity for Sulf and/or to preferential growth.

The plasmid DNA from each Sulf-binding mutant was digested with SpeI/NheI to excise the gene III coding region. The digested plasmids were religated and electroporated into bacteria. Single clones were selected and grown to generate soluble Fab fragments. The soluble Fab were purified from both periplasmic space extracts and culture supernatant using Sulf-BGG-conjugated Sepharose. Using SDS-PAGE, protein bands were observed as 25-kDa doublets (reduced) and as 50-kDa single bands (nonreduced) and were indistinguishable from enzymatically generated 36–65 Fab fragments. The total yield of mutant Fab was 1 to 5 mg/L. Neither induction with IPTG nor incubation at 30°C increased the Fab yield. Amino acid sequence analysis of purified bacterial Fab of wt 36–65 resulted in a mixture of H and L chains in a 1:1 ratio with residues identical to the bona fide hybridoma Ab 36–65. This result indicates that cleavage of the pel B leader occurred at a site (C-terminal to Ala in both heavy and light chain; Fig. 1Go), resulting in chains of native length and with wt N-terminal sequences, indicating that the pComb3 modification was correct.

Binding affinity of selected Sulf-binding clones

Bacterially expressed Fab was purified for each mutant, following gene III excision. Affinity-purified Fab were first tested in a solid-phase direct binding assay against Ars-BGG and Sulf-BGG. The Sulf-selected Fab mutants bound strongly to Sulf-BGG-coated plates, but bound poorly to Ars-BGG-coated plates (Fig. 2Go). Wild-type 36–65 Fab bound Ars-BGG-, but not Sulf-BGG-coated plates, as noted previously (9); the binding of 36–65 Fab produced by enzymatic digestion of IgG and bacterially expressed 36–65 Fab was indistinguishable. The control hybridoma Ab HP Sulf-1 Fab binds Sulf-BGG, but also binds Ars-BGG to a small extent (data not shown) (9). It should be noted that the range of concentrations required to detect binding of 36–65 Fab is higher than that for the corresponding Ig owing to differences in avidity in the solid-phase assay. In summary, the direct binding assays indicated that the selected Fab mutants were specific for Sulf-BGG; all seven new HCDR1 sequences resulted in changing the binding specificity from Ars to Sulf (Fig. 2Go). These seven selected anti-Sulf mutants also did not bind Phos coupled to BGG in a phage ELISA (data not shown).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 2. Direct binding analysis of purified mutant and wt Fab toward Ars-BGG (top) and Sulf-BGG (bottom). Serial fivefold diluted purified Fab fragments (0.64 ng/ml to 50,000 ng/ml) were added to 96-well plates coated with 100 µl of 2 µg/ml Ars-BGG (top) or 2 µg/ml Sulf-BGG (bottom). Binding was detected by ELISA, as described in Materials and Methods. The results for Fab obtained by enzymatic digestion and that produced from bacteria were identical.

 
The binding constants of affinity-purified Fab measured by fluorescence quenching (24, 25) are shown in Table IIGo. Compared with the enzymatically produced HP Sulf-1 Fab (9, 11), five Fab clones, S3, S22, S19, S15, and S23, bound Sulf-N-acetyl-L-tyrosine with affinity up to 8.5-fold higher. All seven selected clones bound Sulf at a level measurable by fluorescence quenching, while the wt 36–65 Ig, Fab produced by enzymatic digestion of 36–65 Ig, and bacterial 36–65 Fab did not bind Sulf (Fig. 2Go).


View this table:
[in this window]
[in a new window]
  		Table II. Binding constants of Abs and mutants

 
Site-directed mutagenesis of HCDR1

Previous mutagenesis experiments indicated that replacement of H:Asn35 by Ala, Ser, Thr, or Cys resulted in Sulf-specific forms of Ab 36–65 (9). The phage-selected Sulf-binding mutants reported in this study exhibit even further diversity at position H:35, i.e., Ile and Val are also observed (Table IGo), albeit in the context of non-wt HCDR1. Replacement of all five residues in 36–65 HCDR1 with glycines (36–65 H1Gly) resulted in complete loss of affinity for Ars (18) (Table IIIGo). However, restoration of the wt residue (Asn) at position 35 (36–65 H1Gly/G35N) resulted in Ars binding (18). Because Asn35 appeared important for Ars specificity, we wondered whether replacement with Ala at position 35 in a polyglycine HCDR1 would result in a shift in fine specificity of the Ab from Ars to Sulf, independent of the identity of other HCDR1 residues. We constructed this mutant as a transfectoma using site-directed mutagenesis. Ab 36–65 H1Gly/G35A bound Sulf, but not Ars, in a direct binding ELISA (Table IIIGo), in contrast to Ab H1Gly/G35N, which is Ars specific. The affinity of Ab H1Gly/G35A for Sulf was 6.3 x 105 M-1 (Table IIIGo). We also assayed the 36–65 mutant containing a polyglycine HCDR1 (36–65 H1Gly) (18), which contains H:Gly35, for Sulf binding. This mutant binds Sulf with an affinity of 1.6 x 105 M-1 (Table IIIGo).

The data reported in this work for phage-selected Fab and site-directed mutants, as well as previous mutagenesis data (9), indicate that at least seven different amino acid replacements at H:Asn35 result in a change in specificity from Ars to Sulf. Furthermore, the results suggest that the specificity change is independent of the context of the remainder of HCDR1. Because all seven phage-selected Sulf mutants contained Gly at position 33, and all Ars-binding canonical Abs also express Gly at this position (30, 31; data summarized in 12, 13), we constructed mutants at H:33 to determine whether other residues were permissive here for Sulf and/or Ars binding. Thus, H:Gly33 was mutated to Ala in both 36–65 and the Sulf-binding mutant Fab S3 (Table IIIGo). The 36–65 G33A mutant showed virtually no binding to Ars-BGG and to goat anti-mouse F(ab')2 in a phage ELISA, indicating poor expression of the mutant in functional form on phage. Control experiments were done to test the integrity of the pComb3 36–65 H G33A construct. The region of plasma DNA encoding the Fd, gene III, and L chain is flanked by two NotI restriction sites in this construct. A NotI restriction digest showed bands of the expected size. A fragment including the entire Fd was obtained by restriction digestion with MunI/SpeI (see Fig. 1Go). The fragment was ligated back into the parental vector that had been predigested with the same enzymes, and the correct placement was verified by DNA sequencing. However, an ELISA on the reengineered mutant plasmid again showed virtually no binding. As bacterial production of this Fab mutant did not appear possible, the mutation was produced instead in hybridoma cells. The mutants 36–65 H:G33A and 36–65 H:G33L were constructed by oligonucleotide-directed mutagenesis, inserted into an expression vector, and electroporated into 36–65 L chain-producing cells (see Materials and Methods). A direct binding G{alpha}M ELISA was used to establish satisfactory expression of these mutants. Direct binding ELISA demonstrated that neither the 36–65 G33L mutant nor the G33A mutant bound Ars or Sulf. These data are summarized in Table IIIGo.

The mutation H:G33A introduced into the Sulf-binding mutant S3 in pComb3, in contrast to that for 36–65, showed expression of Fab using goat anti-mouse F(ab')2 in a phage ELISA. Soluble Fab was produced from the mutant S3 H:G33A following gene III excision and purified by affinity chromatography, and binding assays were performed. A direct binding assay (Table IIIGo) showed that this mutant bound Sulf specifically, but not Ars; the binding was reduced compared with S3. Inhibition ELISA using Sulf-N-acetyl-L-tyrosine as competitor indicated an IC50 approximately 50-fold less than that for wt S3 (data not shown) and no measurable binding in the equilibrium fluorescence-quenching assay (Table IIIGo).

We previously reported that a single mutation in 36–65 at position H:N35 to Ala resulted in Sulf binding (9). To assess the role of Gly33 in such a Sulf-specific mutant Ab, we mutated H:G33 to Ala in a 36–65 Ab containing the D gene junction replacements V100H/Y107S (9, 17) and the H:Asn35 to Ala mutation (9) (Table IIIGo). This Ab binds Ars with higher affinity than 36–65 (9). The junctional residues 100H/107S were observed in Abs elicited in response to both Ars and Sulf (11, 12, 13), and did not affect the specificity dictated by residue H:35 (9). In the direct binding ELISA for the mutant 36–65/V100H/Y107S/N35A/G33A, the binding to Sulf was reduced approximately 100-fold compared with the parent mutant 36–65/V100H/Y107S/N35A (Table IIIGo). Furthermore, this G33A mutant had no measurable affinity using fluorescence quenching.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Immunization of A/J mice with arsonate, phosphonate, or iodoarsonate-protein conjugates results in each case in the selection of canonical Abs associated with the Ars response (10, 32). In contrast, immunization with sulfonate does not elicit Abs using these canonical germline genes. However, when sulfonate was given as a second immunization shortly after primary immunization with Ars, canonical Abs could be recruited that contained somatic mutations resulting in a switch to Sulf binding (11). We previously used site-directed mutagenesis to localize the structural basis for the specificity change (9). Replacement of N35 in H chain CDR1 with Ser, Thr, Ala, or Cys abolished detectable Ars binding in the unmutated canonical Ab 36–65, associated with acquisition of Sulf binding. However, introduction of Gln or Asp into 36–65 at H:35 abolished binding for both Ars and Sulf. Introduction of Ala at H:35 into the somatically mutated higher affinity canonical anti-Ars Ab 36–71 resulted in higher affinity Sulf binding as well (Ka = 3.4 x 107 M-1) with virtual loss of Ars binding. Evidence was adduced using site-directed mutagenesis that the geometries of the hapten contact residues in both 36–65 and 36–71 are similar (20, 28). In the model of the crystal complex of Fab 36–71 with phenylarsonate (12, 13), the side chain of H:Asn35 is a contact residue to Ars. H:Asn35 forms a hydrogen bond with an arsonate oxygen and also is part of a hydrogen bond network with H:Ser99 and H:Trp47. We proposed that the specificity shift to Sulf binding was due to interruption of this hydrogen bond network by replacements at H:35, resulting in a decrease in the binding site size (9). In the new binding site, the putative hydrogen bond geometry was optimal for Sulf as distinct from Phos and Ars. The haptens Ars, Phos, and Sulf differ from each other by only a single atom. The small molecule crystal structures (summarized in 9 show that the maximal difference in solvent-accessible surface is only 14 Å2, and that the bond lengths involving sulfur, phosphorus, or arsenic vary up to a maximum of 0.29 Å. Remarkably, Sulf and Phos are more similar to each other than to Ars, yet Ars and Phos elicit canonical Abs. The pattern of somatic mutations acquired during immunization with Phos is not distinguishable from that of Ars (32). Ab 36–65 and other anti-Ars Abs bind Ars and Phos in a similar manner. It has long been known that (polyclonal) antisera raised to Ars-protein conjugates bound Ars and Phos, but bound poorly to Sulf (33). When mice were immunized first with Ars, then 1 mo later with Sulf, the resulting canonical Abs bound Ars; they also bound Sulf, although generally with lower affinity (10). For the site-directed mutants at H:35 that resulted in Sulf binding, none bound Phos (9). The seven-phage library-derived anti-Sulf Fab mutants described in this study demonstrated the same remarkable specificity difference. In addition, we screened all mutants previously produced in our laboratory in the anti-Ars 36–65 background that were mutated at positions other than H:35, for Ars, Phos, and Sulf binding in direct binding assays (data not shown) (9, 17, 20, 26, 27, 34). All Abs bound Phos as well as Ars, but not Sulf.

Acquisition of Sulf binding is consistent not only with Ala, Thr, Ser, or Cys at position H:35 (9), but in this study we present evidence, based on Sulf selection of phage-displayed HCDR1 mutants, that Val and Ile at this position also confer specificity for Sulf. H:Val35 has been observed independently in Sulf-binding canonical Abs in transgenic mice (L. Wysocki, personal communication). In addition, even Gly at H:35 is associated with Sulf binding, as a mutant 36–65 with polyglycine-substituted HCDR1 binds Sulf (Table IIIGo) (18). Thus, Sulf binding is compatible with at least seven different amino acid side chains at position 35, in contrast to the striking requirement for Asn at H:35 for Ars binding (at least seven different replacements for Asn at H:35 abolish Ars binding) (9, 12, 13, 20, 28). In previous mutagenesis experiments, we showed that Sulf specificity arises from mutations at H:35 in both the germline canonical anti-Ars Ab 36–65 and the somatically mutated anti-Ars canonical Ab 36–71 (the sequence of 36–71 in this region differs from 36–65 only by a Tyr/Asn difference at position 32, a mutation found frequently in other anti-Ars Abs). In addition, we show that Sulf binding can occur in H:35 mutants containing a variety of sequences at other HCDR1 positions (Table IGo). Moreover, Sulf binding also occurs (increased 3.9-fold) when the mutation H:Gly35Ala is introduced into a polyglycine-replaced HCDR1 (Table IIIGo).

The physicochemical conservation at residues 33–36 (Table IGo) in the segment randomized in HCDR1 among Sulf-binding Fab mutants, as well as the conserved residues as defined by Chothia et al. (35, 36) for canonical HCDR1 loop conformation, which are present in 36–65, 36–71, and in the Sulf mutants (residues 26, 27, 29, 34, and 94 in VH), suggests that among this set of Sulf-binding mutants, the HCDR1 loop conformation is not significantly different from that of 36–65 or 36–71. Moreover, it was shown that a polyglycine-substituted HCDR1 in the unmutated canonical Ab does not bind Ars, but can be restored to Ars binding by changing H:35 to the wt Asn (18). Furthermore, mutation of H:Asn35 to Ala in this construct, as noted above, switches fine specificity from Ars to Sulf. These results are consistent with an extended conformation of the HCDR1 loop in the 36–71 crystal structure (12, 13), in which the backbone is relatively solvent inaccessible and rigid, independent of the identity of the side chains. This hypothesis is strengthened by the results of mutagenesis at position H:33 in the CDR1 loop described below. In addition, since 1) among HCDR1 residues, only the side chain of residue 35 is in the binding pocket (Fig. 3Go), and 2) Gly is conserved at position 33 in both Sulf-binding and Ars-binding Abs (see below), the evidence indicates that specificity for both Ars- and Sulf-binding canonical Abs is dictated by the identity of the H:35 side chain. The affinity of Sulf-specific canonical Abs can, however, be modulated by other mutations in HCDR1 (Tables II and III) (11, 25), and was noted previously for Ars-specific canonical Abs (25).



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. Stereoview of the model complex of 36–71 Fab with phenylarsonate (on the left) from the crystal structure (12, 13), including H chain residues 30–36 and HTyr50.

 
Glycine is found at position 33 in the sequences of all seven Sulf clones selected from HCDR1-mutagenized phage-displayed 36–65 Fab (Table IGo). Using the statistical analysis described by Mathews and coworkers (37), the observed frequency of glycine differs from the expected frequency (based on the NNS mutagenesis scheme; see Materials and Methods) by 12.2 SDs. Thus, glycine appears to be highly selected. That glycine is conserved not because it is required for Sulf specificity is indicated by the fact that H:Gly33 is also conserved among more than 227 anti-Ars mAbs utilizing the canonical gene segment combinations that have been sequenced in this region (30, 31, 38; summarized in 12 and 13). We wondered whether H:Gly33 is conserved in canonical Abs binding Ars or Sulf because it is required for proper HCDR1 loop conformation, i.e., because the {phi},{psi} angles at H:33 do not permit substitution of other side chains. However, Ramachandran plots for the crystal structure of the canonical anti-Ars Fab 36–71 (12, 13) indicate that the {phi},{psi} angles of H:33 do not preclude other side chain substitutions at this position. In the 36–71 Fab structure, H:Gly33 is within 5 Å of H:Tyr50, a putative hapten contact residue (Fig. 3Go). Substitutions of residues with longer side chains at H:33 potentially cause steric clash with H:Tyr50, affecting Ars binding. The 36–65 H:Gly33Ala and H:Gly33Leu mutations ablated Ars binding (Table IIIGo). In addition, the mutation H:Gly33Ala reduced Sulf binding substantially in the phage display-derived mutant S3 (Table IIIGo) and in the background of 36–65 containing the specificity-switch mutation Asn35Ala (Table IIIGo). Thus, the mutations at H:33 consistently reduce binding for both Ars and Sulf-specific canonical Abs. These data are in agreement with the conservation of Gly at position 33 in all of the Sulf-binding phage-derived clones reported in this work, as well as all Ars-binding canonical Abs found in vivo. In both in vitro selection by panning and in vivo Ag-driven selection of somatic mutants, selection is based on affinity for Ag.

The side chain at position H:34 in the 36–71 crystal structure packs into the hydrophobic interior (Fig. 3Go), and all of the substitutions at this position among Sulf-binding Fab and other Fab with this particular canonical HCDR1 loop (35, 36) are hydrophobic. At position 36, Trp is invariant among all mouse VH sequences (29); however, Sharon showed that the engineered mutation H:Trp36Ala results in retention of Ars binding and Id in Ab 36–65 (24). Among the Sulf-binding mutants, Phe and Tyr are found at H:36, as well as Trp (Table IGo). The side chain of Trp36 packs into the hydrophobic core in the crystal structure of 36–71; the aromatic residues Phe and Tyr can substitute for Trp, consonant with Sulf binding. One factor that may account for the failure to observe Phe or Tyr at position 36 in vivo is that these two replacements require that more than a single base change occur during somatic mutation.

Because the in vivo hypermutation process resulting in somatic mutations may not be random, the observation that among somatically mutated hybridomas certain residues are conserved cannot be taken as firm evidence that such conservation reflects a required structural and/or functional role. Random mutagenesis of phage-displayed Ab fragments or site-directed mutagenesis provides the potential for changes in specificity and affinity not constrained by the bias of germline gene codons and the in vivo somatic mutation process. On the other hand, there are sequence-specific constraints on expression of Fab in bacterial systems that may not be observed in Abs recruited following somatic cell fusions. Nonetheless, for H chain CDR1 residue 33, the conservation of Gly among anti-Ars and anti-Sulf canonical Abs is related to steric requirements for folding, and the conservation of Asn 35, to arsonate specificity.

Among Abs that display improved affinity following somatic hypermutation and Ag selection, certain point mutations were observed repeatedly, and in some instances were shown to be responsible for the increase in affinity (reviewed in 39 . V region hypermutation does not appear to be random in distribution; not only are there mutational hot spots, but the pattern of nucleotide substitutions is characteristically skewed. Such intrinsic base substitution preferences were used by Betz et al. (39) to distinguish intrinsic mutational hot spots from those that are Ag selected, based on examination of sets of sequences from Ag-selected V genes, as a first step in the elucidation of the (enzymatic) mechanisms of hypermutation. The data reported in this work, in which at least seven amino acid substitutions at the same position significantly increase affinity for sulfonate, suggest that searches for recurrent identical mutations that enhance affinity may fail to identify other unique classes of mutants. Furthermore, the distinction between an intrinsic somatic hypermutation hot spot and a hot spot resulting from selection by Ag (39) cannot necessarily be made if several different amino acid substitutions at the same position can be selected by Ag. The anti-Sulf hybridomas that utilize the Ars-associated canonical set of V genes are to date a small set that includes H:Ser35 substituted for the germline H:35 Asn (11). Whether the other Sulf-selected mutations obtained in vitro occur among in vivo hybridomas is not yet known. It may prove instructive to determine, in a sufficiently large sample, whether certain mutations that are functional in vitro are not observed in vivo. Failure to observe these could be due to 1) a requirement for more than one base change; 2) a locus that is not hypermutable; or 3) the fact that the resultant mutant may be selected against in vivo, as, for example, through autoreactivity.


   Acknowledgments
 
We thank Drs. Carlos Barbas III and Dennis Burton for the pComb3 vector, Drs. Roland K. Strong and Paul Kussie for helpful discussion, Tracy Keel and Rou-Fun Kwong for technical assistance, and Drs. L. Wysocki and J. Sharon for sharing their data. We thank Dr. J. Sharon for providing the polyglycine-HCDR1 cell lines and an expression vector.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant RO1 CA24432 (M.N.M.) and by Cancer Research Institute (D.S.G.). B.P.-S. was supported in part by National Institutes of Health Grant R29 AI33175. Back

2 Address correspondence and reprint requests to Dr. Michael N. Margolies, MGH East, 149 13th Street, Box 31, Charlestown, MA 02129. Back

3 Abbreviations used in this paper: Ars, p-azophenylarsonate; BGG, bovine {gamma}-globulin; Carb, carbenicillin; CDR, complementarity-determining region; G{alpha}M, goat anti-mouse Abs; H chain, heavy chain; HCDR1, heavy chain first complementarity-determining region; HRP, horseradish peroxidase; IdCR, dominant cross-reactive idiotype associated with Abs elicited in response to p-azophenylarsonate; IPTG, isopropyl ß-D-thiogalactoside; L chain, light chain; LB, Luria-Bertani medium; Phos, p-azophenylphosphonate; PVC, polyvinyl chloride; Sulf, p-azophenylsulfonate; Tet, tetracycline; wt, wild-type. Back

4 Amino acids are denoted in a one- or three-letter code in the text. For amino acid mutations, the amino acid preceding the position number represents the unmutated residue. The amino acid following the number is the engineered mutation. Back

Received for publication September 12, 1997. Accepted for publication February 19, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kuettner, M. G., A. L. Wang, A. Nisonoff. 1972. Quantitative investigations of idiotypic antibodies. IV. Idiotypic specificity as a potential genetic marker for the variable regions of mouse immunoglobulin polypeptide chains. J. Exp. Med. 135:579.[Abstract]
  2. Siekevitz, M., S. Y. Huang, M. L. Gefter. 1983. The genetic basis of antibody production: a single heavy chain variable region gene encodes all molecules bearing the dominant anti-arsonate idiotype in the strain A mouse. Eur. J. Immunol. 13:123.[Medline]
  3. Wysocki, L., T. Manser, M. L. Gefter. 1986. Somatic evolution of variable region structures during an immune response. Proc. Natl. Acad. Sci. USA 83:1847.[Abstract/Free Full Text]
  4. Wysocki, L. J., T. Gridley, S. Huang, A. G. Grandea, M. L. Gefter. 1987. Single germline VH and VK genes encode predominating antibody variable regions elicited in strain A mice by immunization with p-azophenylarsonate. J. Exp. Med. 166:1.[Abstract/Free Full Text]
  5. Marshak-Rothstein, A., M. Siekevitz, M. N. Margolies, M. Mudgett-Hunter, M. L. Gefter. 1980. Hybridoma proteins expressing the predominant idiotype of the anti-p-azophenylarsonate response of A/J mice. Proc. Natl. Acad. Sci. USA 77:1120.[Abstract/Free Full Text]
  6. Siekevitz, M., M. L. Gefter, P. Brodeur, R. Riblet, A. Marshak-Rothstein. 1982. The genetic basis of antibody production: the dominant anti-arsonate idiotype response of the strain A mouse. Eur. J. Immunol. 12:1023.[Medline]
  7. Manser, T., L. J. Wysocki, T. Gridley, R. I. Near, M. L. Gefter. 1985. The molecular evolution of the immune response. Immunol. Today 6:94.
  8. Wong, Y. W., P. H. Kussie, B. Parhami-Seren, M. N. Margolies. 1995. Modulation of antibody affinity by an engineered amino acid substitution. J. Immunol. 154:3351.[Abstract]
  9. Kussie, P. H., B. Parhami-Seren, L. J. Wysocki, M. N. Margolies. 1994. A single engineered amino acid substitution changes antibody fine specificity. J. Immunol. 152:146.[Abstract]
  10. Fish, S., E. Zenowich, M. Fleming, T. Manser. 1989. Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response. J. Exp. Med. 170:1191.[Abstract/Free Full Text]
  11. Ellenberger, J., G. Creadon, X. Zhang, L. J. Wysocki. 1993. Recruiting memory B cells with changed antigenic specificity. J. Immunol. 151:5272.[Abstract]
  12. Strong, R. K., R. Campbell, D. R. Rose, G. A. Petsko, J. Sharon, M. N. Margolies. 1991. Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36–71. I. X-ray crystallography, site-directed mutagenesis, and modeling of the complex with hapten. Biochemistry 30:3739.[Medline]
  13. Strong, R. K., G. A. Petsko, J. Sharon, M. N. Margolies. 1991. Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36–71. II. Structural basis of hapten binding and idiotypy. Biochemistry 30:3749.[Medline]
  14. McCafferty, J., A. D. Griffiths, G. Winter, D. J. Chiswell. 1990. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552.[Medline]
  15. Burton, D. R., C. F. Barbas. 1994. Human antibodies from combinatorial libraries. Adv. Immunol. 57:191.[Medline]
  16. Sharon, J., M. L. Gefter, S. L. Morrison, V. T. Oi, M. Ptashne. 1984. Expression of VH C{kappa} chimaeric protein in mouse myeloma cells. Nature 309:364.[Medline]
  17. Parhami-Seren, B., M. N. Margolies. 1996. Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies. J. Immunol. 157:2066.[Abstract]
  18. Sompuram, S. R., W. Den, J. Sharon. 1996. Analysis of antigen binding and idiotypic expression by antibodies with polyglycine-replaced complementarity-determining regions. J. Immunol. 156:1071.[Abstract]
  19. Tabachnick, M., H. Sobotka. 1959. Azoproteins. I. Spectrophotometric studies of amino acid Azo derivatives. J. Biol. Chem. 234:1726.[Free Full Text]
  20. Parhami-Seren, B., P. H. Kussie, R. K. Strong, M. N. Margolies. 1993. Conservation of binding site geometry among p-azophenylarsonate-specific Abs. J. Immunol. 150:1829.[Abstract]
  21. Nisonoff, A.. 1967. Coupling of diazonium compounds to protein. C. A. Williams, and M. W. Chase, eds. In Methods in Immunology and Immunochemistry Vol. 1:120. Academic Press, New York.
  22. II. Barbas, C. F., A. S. Kang, R. A. Lerner, S. J. Benkovic. 1991. Assembly of combinatory antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88:7978.[Abstract/Free Full Text]
  23. Wright, J. F., W. M. Hunter. 1982. A convenient replacement for cyanogen bromide-activated solid phages in immunoradiometric assays. J. Immunol. Methods 48:311.[Medline]
  24. Sharon, J.. 1988. The invariant tryptophan in an H chain V region is not essential to antibody binding. J. Immunol. 140:2666.[Abstract]
  25. Sharon, J.. 1990. Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold. Proc. Natl. Acad. Sci. USA 87:4814.[Abstract/Free Full Text]
  26. Parhami-Seren, B., J. Sharon, M. N. Margolies. 1990. Structural characterization of heavy chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies. J. Immunol. 144:4426.[Abstract]
  27. Parhami-Seren, B., L. J. Wysocki, M. N. Margolies, J. Sharon. 1990. Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype. J. Immunol. 145:2340.[Abstract]
  28. Sompuram, S. R., J. Sharon. 1993. Verification of a model of a F(ab)-complex with phenylarsonate by oligonucleotide-directed mutagenesis. J. Immunol. 150:1822.[Abstract]
  29. Kabat, E. A., M. Reid-Miller, H. M. Perry, K. S. Gottesman. 1991. Sequences of Proteins of Immunological Interest 5th Ed. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, MD.
  30. Manser, T.. 1990. Limits on heavy chain junctional diversity contribute to the recurrence of an antibody variable region. Mol. Immunol. 27:503.[Medline]
  31. Giusti, A. M., T. Manser. 1993. Hypermutation is observed only in antibody H-chain V-region transgenes that have recombined with endogenous immunoglobulin H DNA: implications for the location of cis-acting elements required for somatic mutation. J. Exp. Med. 177:797.[Abstract/Free Full Text]
  32. Fish, S., M. Fleming, J. Sharon, T. Manser. 1991. Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response. J. Exp. Med. 173:665.[Abstract/Free Full Text]
  33. Pressman, D., and A. L. Grossberg. 1973. The Structural Basis of Antibody Specificity. W. A. Benjamin, ed. Advanced Book Program, Reading.
  34. Wong, Y. W., P. H. Kussie, B. Parhami-Seren, M. N. Margolies. 1995. Modulation of antibody affinity by an engineered amino acid substitution. J. Immunol. 154:3351.
  35. Chothia, C., A. M. Lesk. 1987. Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196:901.[Medline]
  36. Chothia, C., A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip, P. M. Colman, S. Spinelli, P. M. Alzari, R. J. Poljak. 1989. Conformations of immunoglobulin hypervariable regions. Nature 342:877.[Medline]
  37. Mathews, D. J., L. J. Goodman, C. M. Gorman, J. A. Wells. 1994. A survey of furin substrate specificity using substrate phage display. Protein Sci. 3:1197.[Abstract]
  38. Vora, K. A., T. Manser. 1995. Altering the antibody repertoire via transgene homologous recombination: evidence for global and clone-autonomous regulation of antigen-driven B cell differentiation. J. Exp. Med. 181:271.[Abstract/Free Full Text]
  39. Betz, A. G., M. S. Neuberger, C. Milstein. 1993. Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes. Immunol. Today 14:405.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Aviszus, X. Zhang, and L. J. Wysocki
Silent Development of Memory Progenitor B Cells
J. Immunol., October 15, 2007; 179(8): 5181 - 5190.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Parhami-Seren, M. Viswanathan, R. K. Strong, and M. N. Margolies
Structural Analysis of Mutants of High-Affinity and Low-Affinity p-Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 2
J. Immunol., November 1, 2001; 167(9): 5129 - 5135.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
M.K. Short, P.D. Jeffrey, A. Demirjian, and M.N. Margolies
A single H:CDR3 residue in the anti-digoxin antibody 26-10 modulates specificity for C16-substituted digoxin analogs
Protein Eng. Des. Sel., April 1, 2001; 14(4): 287 - 296.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, Y. W.
Right arrow Articles by Margolies, M. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, Y. W.
Right arrow Articles by Margolies, M. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS