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Departments of
* Medicine and
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1, 2, 3,
4, and 
1, and the specificity and binding properties of these
mouse-human chimeric (ch) Abs was determined. The chIgG1, chIgG2,
chIgG4, and the chIgA produced annular IF, whereas the IgM and IgG3
produced punctate IF, despite identical V region sequences. Competition
experiments with murine Abs that competed with mAb 18B7 and binding
assays to peptide mimetics of glucuronoxylomannan provided additional
evidence for altered specificity in some of the ch Abs. Expression of
18B7 heavy V region with murine µ C region produced IgM with a
punctate IF, indicating that a change in fine specificity also
accompanied the change from murine IgG1 to IgM. Our results show that
Ab fine specificity can be a function of isotype. This phenomenon may
be most apparent for Abs that bind to Ag with repeating epitopes, such
as polysaccharides, where the quarternary structure of the Ag-Ab
complex may be influenced by such constraints as Fab-Fab angles, Fc-Fc
interactions, Ab size, and solvent accessibility to exposed surfaces.
Alterations in Ab fine specificity following isotype change could have
important implications for current concepts on the generation of
secondary Ab responses to certain Ags and for the isotype preference
observed in Abs to polysaccharides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction of mouse-human chimeric (ch)5 Abs is predicated on the premise that murine V regions confer and retain Ag specificity in the context of expression with human C regions. (2). Mouse-human ch Abs have been used for comparative studies of human C region function (3, 4) and for the calibration of serological assays as human Ab standards (5). Mouse-human ch Abs are more attractive for use in human therapy than murine Abs because they are less immunogenic, have longer serum half-life, and possess human C region effector function (6). Currently, ch Abs are under clinical evaluation for a variety of conditions. The use of mouse-human ch Ab in clinical and laboratory applications is based on the implicit assumption that they retain the specificity of the parent murine Ab.
The murine mAb 18B7 (IgG1-
) is currently in clinical
trial for evaluation as an adjunct to antifungal therapy in the
treatment of Cryptococcus neoformans infections
(7). This mAb binds to glucuronoxylomannan (GXM), which is
the major constituent of the C. neoformans polysaccharide
capsule. To improve effector cell function for human phagocytic cells
and to reduce its immunogenicity in humans, we synthesized ch
human-murine mAbs containing the heavy (VH) and
light (VL) V regions from the murine mAb 18B7 and
human C regions for the various Ig isotypes (µ, 1, 2, 3,
4, and 1; Ref. 3). To our surprise, the 18B7
mouse-human IgM, IgG3, and IgG4 ch Abs revealed differences in fine
specificity from the other ch Abs and the parent murine mAb 18B7
despite identical V region sequences. Similarly, expression of 18B7
VH with murine IgM resulted in altered
specificity. The results imply that in certain conditions, the C region
can affect the conformation of the V region that may in turn lead to
changes in idiotype and fine specificity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IgG1 murine mAb 18B7 has been described (7).
This mAb binds to the C. neoformans polysaccharide and is
currently in clinical evaluation. Cloning of the 18B7
VL regions into pLC and the 18B7
VH region into pHC µ, 1, 2, 3, 4,
and 1 expression vectors was done as described previously
(8). With the exception of IgG3 ch, Abs were expressed in
the murine myeloma cell line NSO as described (8) and
harvested from culture supernatants. Briefly, NSO cells were routinely
grown in DMEM supplemented with 10% FCS and clones stably
transfected with the pLC vector were selected in the presence of 6
µg/ml puromycin. L chain clones stably transfected with the pHC
vectors were selected in the presence of 1.5 mg/ml G418 as described
(8). Cells expressing ch Abs were grown in DME with 10%
FCS, 10% NCTC 109, 1% nonessential amino acids and 1 mg/ml G418 at
37°C and 10% CO2. Subsequently, the cell lines
were adapted to conditions with 5% FCS. For the chIgG3, protein was
obtained by lysis of the transfected cells. The IgM ch Ab was
determined to be pentameric from migration of a nonreducing
SDS-PAGE relative to native human IgM.
Generation of IgG1 switch variant from IgM hybridoma 13F1
An IgG1 isotype variant of the murine mAb 13F1 was recovered by selecting for a spontaneous IgG-producing hybridoma using the ELISA spot method as described (9).
Expression of 18B7 VH with murine IgM
RNA was isolated from hybridoma cells using TRIzol reagent (Life Technologies, Gaithersburg, MD). cDNA was generated using reverse transcriptase and the oligonucleotide primer p(dt)15 (Boehringer Mannheim, Indianapolis, IN). DNA containing the VH was amplified using the primers 5'-ATAAAGCTTGCGGGGATATCCACCATGGAC and 3'-ATAGCTAGCTGAGGAGACTGTGA. The amplified DNA was cloned into the PCR 2.1 vector (Invitrogen, San Diego, CA) for sequencing and subcloned into a murine IgM expression vector, as described (10). The plasmid was then transfected into H chain nonproducing 18B7, 12A1, and 13F1 hybridoma cells by electroporation. Cells were selected with 1.5 mg/ml G418 (Geneticin; Life Technologies) in DME media with 20% of FCS (Harlan, Indianapolis, IN), 10% NCTC-109 (Mediatech, Herndon, VA), and 1% of nonessential amino acid solution (Mediatech).
Immunofluorescence
Glass slides were coated with poly L-lysine (0.1
mg/ml; Sigma-Aldrich, St. Louis, MO), a suspension containing
106 yeast cells was placed on the slide surface
and the slide was air dried. The slides were then blocked by adding a
solution 2% BSA and 0.5% goat serum and incubating for 30 min at
37°C. Abs were then added at a concentration of 5 µg/ml in blocking
solution, and incubated overnight at 4°C. FITC-labeled goat
anti-human IgG, IgM, IgA, or FITC-labeled goat anti-mouse IgM
or IgG1 were added at 10 µg/ml after the incubation with primary Ab,
and incubated at 37°C for 30 min. In other experiments,
tetramethylrhodamine B isothiocyanate-labeled goat anti-human
was used. Prior studies have established that the
immunofluorescence (IF) pattern is the same regardless of whether one
uses FITC-labeled primary or secondary Ab (11). The slides
were washed three times with PBS between application of reagents.
Finally, 30 ml of mounting medium (0.1 M n-propyl
gallate–50% glycerol in PBS; Sigma-Aldrich) was added, and coverslips
were placed. All labeled reagents were from Southern Biotechnology
Associates (Birmingham, AL) except anti-human IgG, which was
obtained from ICN Pharmaceuticals (Costa Mesa, CA). The slides were
viewed with an Olympus AX70 microscope (Olympus, Melville, NY) equipped
with FITC (fluorescein) and tetramethylrhodamine B isothiocyanate
(rhodamine) filters. Fluorescent images were recorded with narrow band
filter sets to ensure no cross-talk or spillover from one filter set to
the others. Gray scale images were merged. This method is equivalent to
multiple exposure photography, but has the advantages of wider linear
response and dynamic range, optical separation of filters, and digital
storage. Images were recorded in black and white. Color corresponding
to the filter wavelength was subsequently added back during image
reconstruction to reflect the actual color observed.
ELISA
mAb concentration was determined by ELISA relative to
isotype-matched standard controls (Sigma-Aldrich). Polystyrene plates
were coated with goat anti-human IgM, IgG, or IgA (Accurate
Chemical and Scientific, Westbury, NY) blocked with 1% BSA in PBS and
incubated with the ch Ab-containing solution. Bound Ab was detected
using alkaline-phosphatase or biotin-conjugated goat anti-human
IgM, IgG, or IgA (Accurate Chemical and Scientific). Ab binding to GXM
was measured as described (6), except that bound Ab was
detected using phosphatase-conjugated goat anti-human (Caltag
Laboratories, San Francisco, CA). Competition ELISAs were done to
determine whether murine mAb 12A1 could inhibit the binding of the ch
Abs to GXM using the same protocols as described previously (7, 8). For this ELISA, polystyrene plates were coated with 2
µg/ml of GXM and incubated for 1.5 h at 37°C. The plates were
then blocked with 1% BSA-PBS and incubated overnight at 4°C.
Ch Abs and mAb18B7 were added and serially diluted, followed
immediately by the addition of IgM mAb 12A1 (final concentration 1
µg/ml). The plates were incubated overnight at 4°C. Binding of
murine IgM was detected by specific alkaline-phosphatase-conjugated
goat anti-mouse reagents. Reactivity of ch Abs with the murine
anti-idiotypic mAb 7B8 was also measured by ELISA. The
anti-idiotypic mAb 7B8 binds to the Ag-combining site of 18B7
(9). Plates were coated with 1 µg/ml of goat
anti-mouse , blocked with 1% BSA in PBS, and incubated with the
ch mAb preparation. The plates were then incubated with 2 µg/ml
solution of mAb 7B8 (IgG1) and binding was detected with
phosphatase-conjugated goat anti-mouse IgG1. For peptide ELISA,
polystyrene plates were coated with 1 µg/ml solution of streptavidin
in PBS followed by blocking with 1% BSA in PBS. Biotinylated peptide
was then added at a concentration of 2 µg/ml before testing mAb
binding by titration. Specific binding of ch18B7 mAb was detected using
phosphatase-conjugated goat anti-human Ab.
To prepare monomers of IgM, the ch mAb (4 µg/ml) was mixed with an equal volume of a 0.15-M solution of 2-ME in TBS and incubated for 1 h at 37°C immediately before addition to the ELISA plate for GXM binding or anti-idiotype ELISAs. The reduced product of IgM and IgG ch Ab contained both H and L chains by ELISA, indicating that the reduction had not disrupted the monomer.
Phagocytosis assay
J774.16 cells were cultured in DMEM with 10% heat-inactivated
FCS, 10% NCTC-109 medium (Life Technologies), and 1% nonessential
amino acids (Cellgro, Washington, DC). The protocol for in vitro
phagocytosis was as described in earlier studies (11) with
minor modifications. Cells were plated in 96-well tissue culture plates
(Falcon; BD Biosciences, Franklin Lake, NJ) at a density of
105 cells/well in 96-well culture plates and
stimulated with 50 U/ml recombinant murine IFN- (Genzyme, Cambridge,
MA) and LPS (Sigma-Aldrich). Briefly, macrophages were stimulated with
50 U/ml IFN-/ml and incubated at 37°C overnight. The medium in
each well was then replaced with fresh medium containing 50 U/ml
IFN- and 1 µg/ml LPS, and C. neoformans cells were
added at a ratio of 5:1 macrophages to fungi. Phagocytosis was measured
in the presence or absence of ch Abs (2 µg/ml). After the addition of
C. neoformans, the cells were incubated at 37°C for 3
h, washed three times with sterile PBS, fixed with cold absolute
methanol, and stained with a 1/20 solution of Giemsa. PBS was used as a
negative control and mAb 18B7 (IgG1) was used as a positive control,
and phagocytic indices were determined. The phagocytic index is defined
by: PI = P x F, where P is
the percentage of phagocytic macrophage and F is the average
of yeast cells per macrophage. Experiments were done in duplicate and
four different fields were counted per well.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 18B7 VH and VL nucleotide sequences have been previously reported based on direct mRNA sequencing (12). The 18B7 VH and VL region cDNA was cloned from 18B7 hybridoma cells by RT-PCR and inserted into human Ig expression vectors as described (8). VH and VL region sequence analysis revealed several differences from the previously published sequences (7, 12). For 18B7 VH, there were three amino acid differences resulting from single-base changes. In the CDR1 region, positions 28 and 29 were Ser and Phe rather than Thr and Cys, respectively, and position 65 of the CDR2 was Arg instead of Lys. For the 18B7 VL, there were two amino acid differences resulting from single-base changes in the cDNA sequence. Amino acid 15 of the first framework region was Leu rather than the original Ala, and CDR1 position 27C was Ala rather than Cys. These updated 18B7 V region sequences are deposited in GenBank under accession nos. AJ309276 and AJ309277. For each cell line producing the ch Abs, the VH and VL regions were sequenced and shown to be identical with each other and with those of the parent mAb 18B7. Multiple clones of each V region were analyzed.
Reactivity of ch Abs with GXM
The IgM, IgG1, IgG2, IgG3, IgG4, and IgA1 mouse-human ch Abs bound
to GXM immobilized in polystyrene plates by ELISA (Fig. 1
). Given that each ch Ab type had
identical V region sequence, we were surprised to find large
differences in binding to GXM (Fig. 1) and to mAb 18B7 (Fig. 1A). The relatively weaker binding observed for the chIgM
was unexpected, since this molecule was pentameric as indicated by
nonreducing SDS-PAGE (data not shown), and the higher valency should
have conferred a higher avidity. To explore the potential contribution
of valence to the binding of the chIgM, 2-ME was used to dissociate the
IgM polymeric structures into monomers (13). Treatment of
IgM with 2-ME resulted in complete loss of Ab reactivity, whereas
identical 2-ME treatment of chIgG1 had no effect on GXM binding (Fig. 1B). Because IgM monomers have the same V region sequence as
the IgG subtypes, the 2-ME reduction experiment indicated that the 18B7
VH-human cµ chimera had significantly reduced
affinity for Ag.
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Prior studies have shown that the IF pattern resulting from Ab
binding to encapsulated C. neoformans cells is a sensitive
indicator of Ab specificity that correlates with biological activity
(11). The IF pattern of mAb 18B7 with C.
neoformans is annular (Fig. 2) and
the expectation was that all the ch derivatives would also produce
annular patterns upon binding yeast cells. The IF pattern observed with
chIgG1, chIgG2, chIgG4, and chIgA1 Abs was annular (Fig. 2).
Nevertheless, we noted differences in the IF pattern for the IgG chs as
manifested by differences in fluorescence intensity throughout the
capsule (Fig. 2). In particular, the chIgG4 gave a thick annular
pattern that was different from the other IgG subtypes. Surprisingly,
the IgM and IgG3 ch Ab produced a punctate pattern that was
qualitatively different from that observed with the parent murine mAb
or the other ch Abs (Fig. 2). The same patterns were obtained
regardless of whether the secondary Ab was specific for the human H or
L chain. The punctate patterns observed with the IgM and IgG3 chs were
similar to that observed with the murine mAb 13F1, which is not
protective and not opsonic (14, 15).
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Because the altered IF patterns for chIgM and chIgG3 suggested an
alteration in V region specificity as a result of expression in
association with human µ and 3, we proceeded to explore this
possibility by Ab competition assays and reactivity with peptide
mimetics. In previous studies, we have used the absence of Ag
competition between two Abs as a criterion for different specificity
(16). Competition experiments were performed with the
murine mAb 12A1 (IgM) that produce annular patterns on binding C.
neoformans and has similar specificity to 18B7 (17).
As is evident in Fig. 3, murine mAb 12A1
inhibited the binding of ch Abs as follows; chIgA1 > chIgG1
> murine 18B7 >> chIgG2 > IgG4 
IgM > IgG3.
Absence of competition with an mAb that inhibits the parent mAb 18B7
provides strong evidence for altered specificity in the chIgM, chIgG2,
chIgG3, and chIgG4 Abs. Competition experiments between mAb 18b7 and ch
Abs revealed that it competed with chIgG1, chIgG2, and chIgG4, but not
chIgM or chIgG3 (data not shown). As a second confirmatory method, we
measured the binding of ch Abs to two GXM peptide mimetics that have
proven useful for identifying small differences in fine specificity
among mAbs with identical V region usage (18). Significant
differences were noted for the reactivity of the various ch Abs and the
parent mAb 18B7 for two peptides, providing additional evidence for
alterations in specificity (Fig. 4).
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The idiotype of mAb 18B7 is recognized by the murine
anti-idiotypic mAb 7B8 (19). The mAb 7B8 interacts
with both the VH and VL at
the Ag combining site (9). The 18B7 Id structure is
presumably conferred by the primary sequence folded into the Ag binding
site. mAb 7B8 bound strongly to chIgG1, chIgG2, and chIgA1 Abs, but
weakly to chIgM, chIgG3, and chIgG4 (Fig. 5). Treating the IgM ch with 2-ME to
create monomers did not restore the 18B7 Id. Hence, there were
significant differences in the reactivity of ch Abs with an anti-Id
mAb to mAb 18B7 despite identical V region sequences.
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The chIgG1, chIgG2, and chIgG4 were strongly opsonic, whereas
chIgA1 had weaker opsonic activity (Fig. 6). The opsonic efficacy of chIgG3 could
not be evaluated because we lacked sufficient protein for this
experiment. The opsonic activity of chIgG1, chIgG2, and chIgG4 was
comparable to that of mAb 18B7. For murine IgM mAbs, the opsonic
activity for C. neoformans correlates with the type of IF
pattern such that Abs with annular IF efficiently promote phagocytosis,
whereas those with punctate IF do not (14, 15). For chIgM,
the opsonic efficacy was significantly lower than the opsonic murine
IgM mAb 12A1, but higher than that observed for the nonopsonic murine
IgM mAb 13F1.
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Because punctate IF patterns had previously been observed only
with murine IgM mAbs (14, 15), we investigated whether
punctate IF was an inherent property of the IgM isotype with certain V
regions by carrying out two experiments. First, we selected a
spontaneous switch variant from the hybridoma 13F1 that synthesized
IgG1. The 13F1 IgG1 variant produced a punctate IF pattern like its
parent IgM (Fig. 2
). Second, we expressed the
18B7VH with a murine µ C region to produce IgM.
We attempted to express the 18B7 VH-µ with
18B7VL, but were unable to obtain an
IgM-producing clone after screening >300 clones in three independent
transfection experiments. Our inability to express 18B7
VH-µ with 18B7VL despite
considerable effort suggests that the isotype switch that produced the
B cell parent for the 18B7 hybridoma resulted in changes that precluded
IgM expression. Instead, we resorted to expressing 18B7
VH-µ with the VL of IgM
mAbs 12A1 and 13F1. Prior studies had established that the IF pattern
is a property of the VH (10). The
18B7 VH-µ was expressed with the
VL of mAbs 12A1 and 13F1, which produce annular
and punctate IF patterns, respectively, as a function of their
VH sequences (10). Binding of 18B7
murine IgM to C. neoformans produced a different IF pattern
than that observed with the parent IgG1 mAb in combination with both
the 12A1 and 13F1 VL (Fig. 2). The 18B7/12A1 and
18B7/13F1 Abs each reacted with GXM, but only 18B7/13F1 was recognized
by the anti-Id mAb 7B8 (Fig. 7).
Competition assays with mAb 18B7 revealed competition with 12A1/13, but
not 18B7/12A1 (Fig. 7). These results imply a change in the fine
specificity of the 18B7 VH when expressed with
murine µ relative to that observed when expressed as 1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that chIgM and chIgG3 gave a punctate IF pattern was intriguing because this type of IF pattern had previously been observed only with two nonprotective IgM murine mAbs (15, 16), which are not opsonic (14). We do not currently know the molecular structure of the carbohydrate domain recognized by the Abs binding in either punctate or annular IF. However, the differences in protective efficacy (15, 16), complement activation (20), and opsonic capacity (14) noted for mAbs that bind in annular in punctate IF patterns provide strong evidence that these represent different epitopes. For this discussion, we use the term "specificity" to denote the binding of Ab to the polysaccharide Ag GXM as manifested by reactivity with GXM, and the term "fine specificity" to denote the nature of Ab binding to GXM as manifested by the interaction of complexes formed by different Abs with GXM that is demonstrated by differences in IF and other serological assays. We are cognizant of the theoretical and practical difficulties involved in defining the terms specificity, fine specificity, and epitope in the absence of structural information (21, 22). Furthermore, we caution that Abs binding in annular or punctate IF are not necessarily binding at exactly the same site on the GXM or forming the same types of Ag-Ab complexes, a notion that finds support in this study.
We investigated whether expression of the 18B7 VH with the human C regions altered Fab structure by evaluating the integrity of the 18B7 Id structure on the ch Abs. The mAb 7B8 demonstrated strong reactivity with chIgG1, chIgG2, and chIgA1, and weaker reactivity with chIgM, chIgG3, and chIgG4. Because the V region sequences of all the chs are identical, this result indicates that expression of the murine 18B7 VH in the context of the human IgM, IgG3, and IgG4 altered the idiotypic structure, possibly a result of differences in protein folding. This observation is consistent with the report that syngeneic V regions are more immunogenic, as IgM molecules when compared with IgG molecules, suggesting isotype-related differences in V region folding and exposure of antigenic epitopes (23).
Reduction of chIgM with 2-ME resulted in complete loss of Ag binding. This was unexpected because IgM monomers should bind to GXM with similar specificity and affinity as the IgG isotypes given identical V region sequences. Because the IgM C region sequence was that expected for this protein (8), mutations or deletions in the cH domains can be excluded as an explanation for the altered fine specificity or the inability to bind as a monomer. Hence, for the chIgM, the change in fine specificity was accompanied by a reduction in apparent affinity and Id recognition consistent with an alteration in the protein structure at the Ag binding site.
Prior studies had shown that the punctate IF fine specificity was conferred by VH (10), but we investigated whether expression of this pattern required the IgM valency structure. The approach was to generate an IgG1 isotype switch variant from a murine IgM that has punctate IF and to express 18B7 VH as a murine IgM. An IgG1 isotype-switched variant was generated from the murine IgM mAb 13F1 that displays punctate IF. Like the parent 13F1 IgM, the 13F1 IgG1 isotype-switched variant revealed punctate IF, a finding indicating that the IF pattern was not restricted to the IgM class. However, expression of the 18B7 VH with the murine µ-chain to generate a murine IgM produced a different IF pattern than the parent IgG1 mAb 18B7. In fact, the pattern of the 18B7 IgM was very similar to the punctate IF observed with chIgM, chIgG3, and mAb 13F1. These results indicate that punctate IF patterns can be observed with both IgM and IgG isotypes. For the specific case of 18B7 IgM, the change from annular to punctate observed for IgG1 and IgM, respectively, may reflect a change in fine specificity resulting from greater avidity. This phenomenon may be similar to that observed to occur for murine IgG3 to streptococcal polysaccharide which can form different types of Ag-Ab complexes than other V region identical IgG isotypes because of ability to aggregate and increase its avidity (1).
chIgG3 also produced a punctate IF pattern, but retained reactivity with the anti-Id mAb, suggesting that steric and/or flexibility constraints may also contribute to the altered fine specificity observed for both IgM and IgG3. Among human IgG subclasses IgG3 is the most flexible, as evidenced by its ability to form ring dimers with anti-Id mAbs (24). IgG3 complexes also demonstrated the largest Fab-Fab angles among IgG subclasses bound to anti-Id mAbs, presumably due to their extended hinge region (24). However, human IgM monomers are even more effective than IgG3 at forming ring dimers, suggesting that it is the most flexible Ab class (25). Because GXM is a polysaccharide with repeating epitopes and both human IgM and IgG3 are multivalent ligands which display great flexibility for their Fab domains, it is possible that the punctate patterns reflect the formation of Ag-Ab complexes in the capsule with different geometry than those associated with the annular IF pattern. Consistent with this view is the observation that Abs which produce annular IF on wild-type C. neoformans strains produce punctate IF when binding to mutants that express O-acetylation-deficient GXM, which is more flexible polysaccharide structure and may form different types of Ag-Ab complexes in the capsule (26). Another factor that may contribute to the type of complexes formed is the nature of the exposed surfaces of the various isotypes. In this regard, liquid partition studies have shown that human IgM and IgG3 also appear to have different surface properties from each other and from other IgG subclasses which could conceivably affect the microenvironment in which the complexes are formed (27). For chIgG4, the alteration of the IF pattern to a thick annular type may reflect the ability of IgG4 molecules to self interact, a phenomenon proposed to account for both its apparent monovalence and its capacity to form bispecific molecules (28).
In summary, we observed a change in IF pattern from annular to punctate
when 18B7 VH was expressed with murine and human
µ C region and with human 3 c and 4 c. For each Ab, the
explanation for the change in fine specificity may be different. For
chIgM, this phenomenon may reflect an altered Fab structure (as
evidenced by loss of Id reactivity and reduced affinity), greater
intrinsic Fab flexibility (25), or different Ig surface
properties (27). For the murine 18B7 IgM, the change in
specificity may reflect a difference in avidity as reported for Abs to
streptococcal polysaccharide (1). For the chIgG3, the
change in specificity may reflect the enhanced Fab flexibility of this
isotype relative to other IgGs (24), or different Ig
surface properties (27). Furthermore, we did note
variations in IF after binding C. neoformans and in the
reactivity with GXM and peptide mimetics for the IgG1, IgG2, and IgG4
chs, suggesting that more subtle changes in fine specificity may have
also occurred in the other ch Abs.
The results of this study also shed light on the requirements for generation of the punctate IF pattern. Before this study, the punctate IF pattern had been observed only with two murine IgMs known as 13F1 and 21D2 (15). Recently, we used site-directed mutagenesis to establish that changing two amino acids in the in V region of an IgM mAb could change the IF pattern from annular to punctate (10). In this study, we demonstrate that the same V can produce annular or punctate patterns depending on which human or mouse C region it is associated with. Hence, the type of IF pattern is dependent on both the primary structure of the V region and the conformation of the Ag combining sites as determined by the associated C region.
Our observations add to the growing body of evidence that in certain circumstances, the Ig C region can affect the fine specificity of an Ab molecule. There are at least two potential mechanisms for this phenomenon. First, the C region could affect V region folding. This mechanism could explain isotype-related differences in Id immunogenicity (23) and the simultaneous loss of Id recognition for the chIgM and altered IF pattern on binding C. neoformans noted in this study. Second, the C region could influence the specificity of an Ab for an Ag with repeating epitopes through effects on segmental flexibility and the ability of certain Fc regions to associate (1). This phenomenon was demonstrated for a family of murine mAbs to group A streptococcal polysaccharide with identical V region which show isotype-related differences in their ability to bind bacterial strains expressing different epitope densities (1). Furthermore, there is evidence that C regions can affect the neutralizing activity of V-region identical Abs to human cytomegalovirus, possibly as a result of differences in the binding domain of the Ab to the target glycoprotein as a consequence of Fc-related effects (29). Another study reported that changing the C region of a viral enhancing murine mAb from IgG3 to a human IgG1 abrogated its deleterious activity in vitro, possibly as a result of changing the three-dimensional structure of the virus-Ab complex (30).
The observation that the C region can affect the specificity of an Ab raises the possibility that isotype switching in the immune response can be accompanied by a change in specificity. If this is the case, then isotype switching may lead to loss of recognition of the original Ag as well as the recognition of new epitopes. In fact, this could suggest an explanation for the observation that the primary and secondary response to certain Ags can arise from different precursors (31). A variation of this theme was suggested by the observation that IgA1 bind tubulin with significantly greater affinity than IgG1 as a consequence of cH segmental flexibility, raising the possibility that affinity maturation can occur by isotype switching alone (32). Furthermore, our findings imply that certain types of Ag-Ab interactions may be limited to certain C regions and suggest a potential explanation for the biased preference of certain isotypes in polysaccharide responses. From a practical consideration, our observations suggest that fine specificity changes may be a relatively common phenomenon when ch or humanized Abs are constructed. This phenomenon may be limited to certain murine V regions, and/or may apply primarily to V regions that bind to Ags with repeating epitopes such as polysaccharides. Furthermore, the results are consistent with the view that differences in hinge angle and flexibility, possibly in combination with other variables such as C region size and glycosylation can affect the type of Ag-Ab complexes made. Our findings provide additional validation for the theory proposed by N. S. Greenspan (33) that valency can influence the specificity of the Ag-Ab interaction. Given the increasing use of mouse-human ch Abs in human therapy, it is critical that each ch Ab be carefully tested for specificity to insure the fidelity of the Ab engineering process.
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Acknowledgments |
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Footnotes |
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2 G.R.M. and M.T. contributed equally to this study.
3 Current address: Biomedical Research Center, University of British Columbia, Vancouver, British Columbia, Canada.
4 Address correspondence and reprint requests to Dr. Arturo Casadevall, Albert Einstein College of Medicine, 1300 Morris Park Avenue G701, Bronx, NY 10461. E-mail address: casadeva{at}aecom.yu.edu
5 Abbreviations used in this paper: ch, chimeric; VH, heavy V region; VL, light V region; GXM, glucuronoxylomannan; IF, immunofluorescence.
Received for publication January 29, 2002. Accepted for publication May 15, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. J. LaRocca, L. I. Katona, D. G. Thanassi, and J. L. Benach Bactericidal Action of a Complement-Independent Antibody against Relapsing Fever Borrelia Resides in Its Variable Region J. Immunol., May 1, 2008; 180(9): 6222 - 6228. [Abstract] [Full Text] [PDF]
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 G. Li, S. Andreansky, G. Helguera, M. Sepassi, N. Janikashvili, J. Cantrell, C. L. LaCasse, N. Larmonier, M. L. Penichet, and E. Katsanis A chaperone protein-enriched tumor cell lysate vaccine generates protective humoral immunity in a mouse breast cancer model Mol. Cancer Ther., March 1, 2008; 7(3): 721 - 729. [Abstract] [Full Text] [PDF]
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 O. Zaragoza, M. Alvarez, A. Telzak, J. Rivera, and A. Casadevall The Relative Susceptibility of Mouse Strains to Pulmonary Cryptococcus neoformans Infection Is Associated with Pleiotropic Differences in the Immune Response Infect. Immun., June 1, 2007; 75(6): 2729 - 2739. [Abstract] [Full Text] [PDF]
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M. Torres, N. Fernandez-Fuentes, A. Fiser, and A. Casadevall The Immunoglobulin Heavy Chain Constant Region Affects Kinetic and Thermodynamic Parameters of Antibody Variable Region Interactions with Antigen J. Biol. Chem., May 4, 2007; 282(18): 13917 - 13927. [Abstract] [Full Text] [PDF]
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D. O. Beenhouwer, E. M. Yoo, C.-W. Lai, M. A. Rocha, and S. L. Morrison Human Immunoglobulin G2 (IgG2) and IgG4, but Not IgG1 or IgG3, Protect Mice against Cryptococcus neoformans Infection Infect. Immun., March 1, 2007; 75(3): 1424 - 1435. [Abstract] [Full Text] [PDF]
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 S. Rafati, N. Hassani, Y. Taslimi, H. Movassagh, A. Rochette, and B. Papadopoulou Amastin Peptide-Binding Antibodies as Biomarkers of Active Human Visceral Leishmaniasis Clin. Vaccine Immunol., October 1, 2006; 13(10): 1104 - 1110. [Abstract] [Full Text] [PDF]
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 R. A. Larsen, P. G. Pappas, J. Perfect, J. A. Aberg, A. Casadevall, G. A. Cloud, R. James, S. Filler, and W. E. Dismukes Phase I Evaluation of the Safety and Pharmacokinetics of Murine-Derived Anticryptococcal Antibody 18B7 in Subjects with Treated Cryptococcal Meningitis Antimicrob. Agents Chemother., March 1, 2005; 49(3): 952 - 958. [Abstract] [Full Text] [PDF]
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M. Torres, R. May, M. D. Scharff, and A. Casadevall Variable-Region-Identical Antibodies Differing in Isotype Demonstrate Differences in Fine Specificity and Idiotype J. Immunol., February 15, 2005; 174(4): 2132 - 2142. [Abstract] [Full Text] [PDF]
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U. K. Buchwald, A. Lees, M. Steinitz, and L.-a. Pirofski A Peptide Mimotope of Type 8 Pneumococcal Capsular Polysaccharide Induces a Protective Immune Response in Mice Infect. Immun., January 1, 2005; 73(1): 325 - 333. [Abstract] [Full Text] [PDF]
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M. W. Oli, N. Rhodin, W. P. McArthur, and L. J. Brady Redirecting the Humoral Immune Response against Streptococcus mutans Antigen P1 with Monoclonal Antibodies Infect. Immun., December 1, 2004; 72(12): 6951 - 6960. [Abstract] [Full Text] [PDF]
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R. W. Maitta, K. Datta, Q. Chang, R. X. Luo, B. Witover, K. Subramaniam, and L.-a. Pirofski Protective and Nonprotective Human Immunoglobulin M Monoclonal Antibodies to Cryptococcus neoformans Glucuronoxylomannan Manifest Different Specificities and Gene Use Profiles Infect. Immun., August 1, 2004; 72(8): 4810 - 4818. [Abstract] [Full Text] [PDF]
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M. D. Meeks, R. Saksena, X. Ma, T. K. Wade, R. K. Taylor, P. Kovac, and W. F. Wade Synthetic Fragments of Vibrio cholerae O1 Inaba O-Specific Polysaccharide Bound to a Protein Carrier Are Immunogenic in Mice but Do Not Induce Protective Antibodies Infect. Immun., July 1, 2004; 72(7): 4090 - 4101. [Abstract] [Full Text] [PDF]
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 J. P. Pandey, J. Astemborski, and D. L. Thomas Epistatic Effects of Immunoglobulin GM and KM Allotypes on Outcome of Infection with Hepatitis C Virus J. Virol., May 1, 2004; 78(9): 4561 - 4565. [Abstract] [Full Text] [PDF]
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D. C. McFadden and A. Casadevall Unexpected Diversity in the Fine Specificity of Monoclonal Antibodies That Use the Same V Region Gene to Glucuronoxylomannan of Cryptococcus neoformans J. Immunol., March 15, 2004; 172(6): 3670 - 3677. [Abstract] [Full Text] [PDF]
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R. W. Maitta, K. Datta, A. Lees, S. S. Belouski, and L.-a. Pirofski Immunogenicity and Efficacy of Cryptococcus neoformans Capsular Polysaccharide Glucuronoxylomannan Peptide Mimotope-Protein Conjugates in Human Immunoglobulin Transgenic Mice Infect. Immun., January 1, 2004; 72(1): 196 - 208. [Abstract] [Full Text] [PDF]
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T. Burns, Z. Zhong, M. Steinitz, and L.-a. Pirofski Modulation of Polymorphonuclear Cell Interleukin-8 Secretion by Human Monoclonal Antibodies to Type 8 Pneumococcal Capsular Polysaccharide Infect. Immun., December 1, 2003; 71(12): 6775 - 6783. [Abstract] [Full Text] [PDF]
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T. E. Michaelsen, O. Ihle, K. J. Beckstrom, T. K. Herstad, J. Kolberg, E. A. Hoiby, and A. Aase Construction and Functional Activities of Chimeric Mouse-Human Immunoglobulin G and Immunoglobulin M Antibodies against the Neisseria meningitidis PorA P1.7 and P1.16 Epitopes Infect. Immun., October 1, 2003; 71(10): 5714 - 5723. [Abstract] [Full Text] [PDF]
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