(Received for publication, November 28, 1995; and in revised form, December 28, 1995)
From the
Recombinant Fv derivative of the high affinity murine
anti-fluorescein monoclonal antibody 4-4-20 was constructed and
expressed in high yields, relative to the single chain antibody (SCA)
derivative (2-3-fold), in Escherichia coli. Both
variable heavy (V) and variable light (V
)
domains, that accumulated as insoluble inclusion bodies, were isolated,
denatured, mixed, refolded, and affinity-purified to yield active Fv
4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding
properties compared with the SCA construct, both were slightly lower
than the affinities expressed by Fab or IgG 4-4-20. Proper protein
folding was shown to be domain-independent by in vitro mixing
of individually refolded variable domains to yield functional Fv
protein. In solid phase and solution phase assays, Fv 4-4-20 closely
approximated the SCA derivative in terms of both idiotype and metatype,
confirming identical active site structures and conformations. The
equilibrium dissociation constant (K
) for
the V
/V
association (1.43
10
M), which was determined using the
change in fluorescein spectral properties upon ligand binding, was
relatively low considering the high affinity displayed by the Fv
protein for fluorescein (K
, 2.9
10
M). Thus, domain-domain stability in
the Fv and SCA 4-4-20 proteins cannot be the sole cause of reduced
affinity (2-3-fold) for fluorescein as compared with the Fab or
IgG form of 4-4-20. With their identical ligand binding and structural
properties, the decreased SCA or Fv affinity for fluorescein must be an
ultimate consequence of deletion of the C
1 and C
constant domains. Collectively, these results verify the
importance of constant domain interactions in antibody variable domain
structure-function analyses and future antibody engineering endeavors.
Antibody Fv fragments are composed of heavy chain
(V) (
)and light chain (V
) variable
domains. These two domains associate noncovalently to form the smallest
functional antibody protein capable of antigen binding that most
closely approximates the Ig molecule(1, 2) . These
proteins have been previously found to be less stable in terms of
domain-domain association than Fab fragments due to the lack of
covalent bonds between the two variable
domains(3, 4) . Single chain antibody (SCA) molecules
have been produced to diminish this instability by the introduction of
an interdomain linker peptide(5, 6) . SCA proteins
often mimic the parent antibody active site in terms of antigen binding
and structural properties with usually some reduction in affinity for
antigen(7, 8, 9) . Recently, Fv proteins have
been engineered to possess an interdomain disulfide linkage,
effectively disallowing dissociation of the two domains(10) .
Due to their small size and amenability to genetic engineering,
recombinant Fv proteins have been widely applied in the study of
antibody active site
structure-function(7, 11, 12, 13) ,
idiotypy and
metatypy(14, 15, 16, 17) , antibody
bivalency and
bispecificity(18, 19, 20, 21) , and in vivo immunodiagnostics and
therapy(10, 22, 23) .
Fv molecules have
been efficacious proteins in the study of antibody active site
structure-function and protein stability. Studies involving comparative
analysis of Fv protein with other immunoglobulin constructs afford
unique opportunities for determining domain-domain interactions and the
effects these interactions exert upon the intrinsic conformational and
antigen binding properties of the variable domains. Being covalently
coupled by an interdomain linker, SCA proteins have been suggested to
possess greater interdomain stability than their Fv counterparts due to
the favorable entropic effect of domain coupling (6, 8) . This would, in turn, suggest that in the
appropriate Fv molecule (one with high affinity for antigen),
interdomain associative properties would dictate the overall affinity
displayed for antigen because only associated V/V
proteins would bind antigen. In previous studies, dissociation
constants for the V
/V
association in Fv
molecules varied from 10
to >10
M(3, 24, 25, 26) .
These Fv molecules also displayed similar dissociation constants for
their respective antigens (10
to >10
M), further supporting some correlation between
interdomain and active site/antigen interactions. Further analysis of
V
/V
association constants in relation to
antigen affinity would allow identification of components necessary for
the production of stable Fv molecules and novel variable domain
proteins.
Fv molecules have been especially useful in the study of
idiotypy and metatypy. Antibody idiotype and metatype are
immunologically resolved markers of active site structural and
conformational determinants in the unliganded and liganded state,
respectively (review in Refs. 27 and 28). Indeed, the transition
between the idiotypic and metatypic states upon ligand binding
emphasizes the dynamic properties of antibody proteins(15) .
The principle of antibody dynamics is governed by the process of
structural fluctuation in both the unliganded and liganded states until
the most energetically favorable state is
established(29, 30) . Thus, understanding how
structural and dynamic features are exerted within antibody variable
domains will directly influence our understanding of antibody active
site/ligand binding properties. Small differences in antigen binding
affinities have been found between certain SCA and IgG despite their
structural relatedness(7) . The possibility exists that
constant/variable domain interactions results in more than structural
rigidity, but are responsible for restricted variable domain
conformational dynamics favoring antigen binding. Indeed, studies with
MOPC 315, a murine anti-nitrophenyl IgA antibody, have indicated that
the C1 domain in Fab fragments influences idiotypic
expression of the antibody through a dynamic effect on the variable
domain structure(31) . In other antibodies, studies have
demonstrated the importance of cooperative interface interactions (cis
and trans) between the variable and constant domains regarding variable
domain stability and antigen binding(7, 32) . To
examine this, Fv and SCA proteins, due to differences in interdomain
associative properties with similar ligand binding to IgG, represent
critical reagents to study ligand binding properties and variable
region structural features as influenced by antibody constant domains.
To study the relation between interdomain association and affinity
for antigen, an antibody protein must be available in many derivative
forms (e.g. with and without constant domain, with and without
an intervariable domain linker). To satisfy these criteria, the Fv
derivative of mAb 4-4-20, a high affinity murine anti-fluorescein
antibody, has been synthesized. mAb 4-4-20 was a suitable antibody for
this study due to its high degree of structural characterization (7, 33, 34) and the previous construction and
characterization of SCA 4-4-20(7, 35) . SCA 4-4-20 has
been studied extensively in terms of active site
environment(36, 37) , antigen binding
structure(13) , and thermodynamics(8, 38) .
Comparison of SCA with Fab 4-4-20 showed almost identical
guanidine-induced denaturation profiles, idiotype and metatype
expression, yet a 2-3-fold reduction in affinity for
fluorescein(7) . The structural and antigen binding properties
required for fluorescein binding and quenching by SCA and mAb 4-4-20
have also been extensively analyzed in relation to the remainder of the
4-4-20 idiotype anti-fluorescein antibody family(11) .
Comparison of Fv 4-4-20 with SCA, Fab, and IgG may assist in
understanding the basis for this difference in affinity for antigen
upon removal of the 4-4-20 constant domains. Studies reported here show
that Fv 4-4-20 possessed identical structural, idiotypic, metatypic,
and ligand binding properties as SCA 4-4-20. With confirmation of
identical ligand binding and structural characteristics between Fv and
SCA, the dissociation constant (K) for
the V
/V
association was determined and analyzed
in relation to the Fv affinity for fluorescein. Such analyses
implicated the necessity of constant domain/variable domain association
in the formation of the high affinity liganded state. Overall, results
indicated that the reduced affinity of Fv and SCA 4-4-20 did not
correlate with reduced variable domain association, but with the
absence of antibody constant domains, emphasizing their role in
antibody/antigen interactions.
Figure 1:
Polynucleotide primers and
amplification strategy used for construction of the V and
V
gene products. Regions of complementarity with 4-4-20 are underlined. OmpA and terminator complementary sequences are in italics, and vector complementary sequences are roman.
Following amplification, V and V
PCR
products were purified in low melting temperature agarose (Seaplaque,
FMC) and cloned into SmaI digested pTZ18u(48) .
Correct clones were identified by restriction length analysis and
verified by dideoxy sequencing. To construct the V
gene,
PCR was used to add the transcription stop codons by 3` primer overhang (Fig. 1). To incorporate the signal sequence in the V
gene, PCR was used to amplify the OmpA sequence with the addition
of the V
5` sequence to the 3` end of OmpA. The resulting
PCR product was then used as the 5` primer to incorporate the signal
sequence to the V
gene (Fig. 1). Both amplified
genes were cloned into pTZ18u to form pJWc2-2 and pJWc1-5,
respectively. Following verification of proper primary sequence, the
V
and V
genes were excised using ClaI-BamHI and cloned into pGX8773 for expression.
where Q is the ratio between quantum yields of free and
bound ligand, r is the anisotropy value for
fluorescein-labeled variable domain (0.02), and r
is the anisotropy for totally bound fluorescein (0.32). Protein
concentrations were corrected for the R value of Fv affinity
labeling.
Figure 2:
Solid phase ELISA analysis of Fv 4-4-20. A, direct binding of IgG, SCA, or Fv 4-4-20 (10 µg/ml
starting concentration) to fluorescein-BSA-coated wells. Protein was
detected with mAb 3A5-1 (10 µg/ml) and horseradish
peroxidase-anti-hamster IgG. B, 4-4-20 proteins
(10 to 10
M) were used
to inhibit anti-metatype antibodies (10 ng/ml) from binding to wells
coated with affinity-labeled Fab 4-4-20. C, 4-4-20 proteins
(10
to 10
M) were used
to inhibit horseradish peroxidase-IgG 4-4-20 (10 ng/ml) from binding to
wells coated with anti-idiotype antibodies. Individual points represent
mean values of triplicate trials with standard deviations (error
bars). Points lacking error bars indicate standard deviations
smaller than symbol.
Figure 3:
Fluorescein fluorescence quenching
comparison of purified Fv 4-4-20 protein with mixed V and
V
protein. Domains were mixed so that the starting
concentration of Fv and mixed variable domains would be approximately
equivalent. Individual points represent mean values of triplicate
trials.
Anti-fluorescein
antibodies also produce a characteristic bathochromic shift of
10-20 nm in the ligands absorption maximum ()
upon fluorescein binding(40, 64) . The bathochromic
shift in bound fluorescein absorption was identical for SCA and Fv
4-4-20 (504 nm) (Table 1).
Figure 4:
CD
spectra of IgG and Fv 4-4-20 (both 0.5 OD units). At
the concentration indicated (>5 µM), the Fv sample
should be in the associated form.
Figure 5: Pressure-induced dissociation of fluorescein profiles for Fv and SCA 4-4-20. Equal molar samples (0.127 µM) of protein were subjected to increasing hydrostatic pressure. Total fluorescein fluorescence intensity values were recovered at each pressure and compared with free fluorescein fluorescence intensity values.
Figure 6:
Dissociation of Fv 4-4-20 domains by
serial dilution as detected by fluorescein fluorescence.
Affinity-liganded protein was serially diluted and fluorescein
anisotropy measured at each protein concentration value (domain
association being concentration-dependent). Excitation wavelength at
480 nm and emission wavelength at 530 nm. Points represent mean values
of triplicate trials. Recovered K = 1.43 (± 0.17)
10
M.
In terms of structure-function relationships, recombinant Fv
proteins have been invaluable tools for experimental studies of
immunoglobulins. More recent endeavors involving these recombinant
proteins have included their engineering with specialized effector
functions for in vitro and in vivo immunodiagnostic
and therapeutic roles. A common characteristic upon production of these
diminutive antibody proteins is that their affinity for antigen is
often reduced (or abrogated) as compared with the parental IgG. The
reduced affinity has been attributed to changes in the active site
structure or variable domain associative properties upon removal of the
constant domains(3) . If the initial decrease in Fv affinity
for antigen was due to decreased domain-domain interactions, the
properties governing stable variable domain association in relation to
antigen binding must be identified. As such antibody proteins continue
to be modified and applied to different systems (reviewed in (67) and (68) ), the nature of this affinity decrease,
including how V/V
affinity correlates with
antigen binding affinity, must be defined and exploited. The well
characterized 4-4-20/fluorescein system presented an ideal method to
study this phenomenon, based on the fact that SCA 4-4-20 exhibits a
slight decrease in affinity for antigen compared with IgG(7) .
This study addressed the question by production and characterization of
the Fv analogue of the 4-4-20 active site. These studies were based on
the premise that comparative analysis provided clarification of the
correlation between antibody constant domains, variable domain
stability, and affinity for antigen.
Using similar expression
conditions for SCA, purified Fv 4-4-20 demonstrated nearly identical
anti-fluorescein activity as SCA (Fig. 2A).
Polyacrylamide gel analyses confirmed that the purified Fv protein
contained only V and V
domain proteins (data
not shown). In terms of expression yield, E. coli cultures
producing V
and V
protein consistently yielded
2-3-fold more active Fv protein than similar cultures producing
SCA upon refolding and affinity purification. The fact that improper
disulfide bonds could not form between variable domain proteins during
expression and refolding was most likely responsible for this
result(4) . In terms of idiotypy and metatypy, Fv 4-4-20 showed
properties identical to SCA 4-4-20 when examined with polyclonal 4-4-20
variable domain-specific antibodies (Fig. 2, B and C). These results suggested that despite the dependence on
noncovalent interactions for association, Fv 4-4-20 closely
approximated the SCA molecule in terms of unliganded and liganded state
structure.
Ligand binding affinities and ligand-related spectral
measurements were made to assess Fv homology to the SCA molecule (in
terms of the initial decrease in affinity for antigen). Such spectral
measurements involving fluorescein/anti-fluorescein antibodies are
characteristic of the specific anti-fluorescein active site environment
which are relatively independent of affinity(69) . Fv 4-4-20
showed almost identical ligand-related spectral properties (Q and
) and affinity for
antigen relative to SCA (Table 1). Anti-metatype antibodies, both
polyclonal and monoclonal, characteristically enhance the affinity for
fluorescein displayed by the anti-fluorescein active site for which
they are specific(15, 54) . Fluorescein affinity
measurements were repeated for Fv and SCA 4-4-20 in the presence of
anti-metatype reagents to assess their relationship in terms of ligand
binding kinetics and liganded state conformation. Fv and SCA showed
similar (proportional) increases in affinity in the presence of
anti-metatype antibodies, confirming that both active site structures
possess the same conformational perturbations upon ligand binding (Table 1).
In addition, CD analysis suggested identical
overall secondary structures for Fv and SCA 4-4-20. Fv 4-4-20 showed
the identical positive (204 nm) and negative extremum (217 and 230 nm)
as SCA(36) . The negative extrema at 217 nm with a shoulder
near 230 nm, found characteristically in immunoglobulin CD spectra, are
typical of proteins with -sheet structure and a high aromatic
content(70, 71) (i.e. SCA 4-4-20).
Interestingly, the CD spectra of isolated variable domains consisted of
negative extrema at 217 nm, indicative of
-sheet structure, but
also showed negative values at 204 nm, possibly due to a higher degree
of random structure (65) (data not shown). The shoulder at 230
nm in the CD spectra of both V
and V
proteins
was reduced compared with the Fv, suggesting a possible re-orientation
of tryptophan and tyrosine side chains in their respective
environments(37) . This would indicate that isolated domain
proteins undergo dynamic secondary structure rearrangement in order to
dimerize and form active Fv protein. To support this result,
comparative fluorescein quenching studies were performed using
affinity-purified Fv and associated V
and V
proteins. Associated protein showed almost identical fluorescein
quenching properties as compared with an equal optical density solution
of Fv (Fig. 3). It was also demonstrated that the liganded
V
/V
dimers responded similarly to
affinity-purified Fv when affinity measurements were determined in the
presence of anti-metatype reagents (data not shown). Collectively,
these results indicated that 1) Fv, V
, and V
proteins consisted of mostly
-sheet structure and some
random coil, 2) upon V
and V
association some
conformational changes are necessary for proper dimerization and active
site formation, and 3) individually refolded domains maintain a
dimerization competent form in the absence of constant domains which
can form the proper active site environment for fluorescein binding and
quenching.
As previously stated, hydrostatic pressure does not
promote changes on the tertiary structure of proteins, but alters
regions of secondary structure responsible for global protein
conformation(38, 66) . A comparison of the pressure
induced dissociation of fluorescein profiles for Fv and SCA would be a
definitive evaluation of their dynamic similarity. Identical
fluorescein fluorescence profiles were recovered for the two proteins
when exposed to increasing hydrostatic pressure (Fig. 5). This
indicated that Fv 4-4-20 displayed the same standard volume change
(: -50 ml/mol) upon fluorescein dissociation
as SCA(38) . Seeing that their structures were apparently
identical, this suggested that the Fv 4-4-20 must have increased
conformational dynamics relative to the IgG molecule
(
: -5 ml/mol) as originally postulated for
SCA(15, 38, 41) . This indicated that
increased dynamics were responsible for the decreased affinity for
antigen displayed by Fv and SCA. Determination of the Fv interdomain
dissociation constant (1.43
10
M)
showed that despite the relatively low associative affinity, the high
affinity fluorescein interaction was unchanged relative to the SCA (Fig. 6). This excluded the possibility that the initial
decrease in the affinity for fluorescein upon removal of the constant
domains was due to decreased domain-domain stability. The large
difference between V
/V
and Fv/fluorescein K
values (
400-fold) suggested that in terms
of 4-4-20, there was little or no quantitative correlation between
interdomain stability and antigen affinity. Seeing that individual
variable domain proteins showed no affinity for antigen (data not
shown), this confirmed that there was no coupling of fluorescein
binding or domain association free energy in the formation of the Fv
4-4-20(72) . Thus, Fv structural characteristics responsible
for interdomain association were independent of the structural features
necessary for high affinity antigen binding. Collectively, results
indicated that the absence of constant domains caused increased dynamic
flexibility, not reduced variable domain associative affinity, in Fv
and SCA 4-4-20 and resulted in decreased affinity for antigen.
Previous studies have demonstrated that heavy chain isotype (i.e. constant domain structure) influences antibody functional affinity against multivalent antigen(73, 74) . The effect of constant domains reported in these studies, which depended on high multivalent antigen concentrations, suggested that the change in functional affinity was due to change in segmental flexibility of the IgG molecule. Antibody isotype was, however, implicated in the expression of idiotopes on the variable domains of an anti-nitrophenyl antibody MOPC 315(31) . Idiotopes represent structural markers on the antibody active site which are sensitive to conformational fluctuations due to either ligand binding or natural protein dynamics(29, 30, 75) . Such relationships would support the hypothesis that the interaction between the variable and first constant domains are necessary for proper variable domain conformational dynamics and not rigid structural features (Fig. 7). Results presented here support this hypothesis by demonstrating how the absence of constant domains influences active site/antigen interactions. In the case of 4-4-20, the binding of fluorescein can be considered a perturbation of the active site conformation which the constant domains can restrict to maintain the high affinity interaction. Removal of the constant domains from the SCA and Fv constructs resulted in the removal of this ``dynamic buffering'' effect. The ensuing increased domain dynamics translated into an increased dissociation rate of fluorescein from the active site. As studies progress on the re-engineering of antibody proteins, care must be taken to assess the importance of constant domain interactions for proper variable domain function. Methods which can both stabilize the active site structure and maintain wild type conformational dynamics may be necessary to ensure the success in producing recombinant Fv proteins which mimic parental IgG affinities.
Figure 7: Diagram of the domain-domain interactions required for proper variable domain dynamics. Cis interactions represent those interactions which involve contacts and dynamics in the vertical plane. Trans interactions represent those which involve contacts and dynamics in the horizontal plane (both C-C and V-C domain interactions).