(Received for publication, January 19, 1996; and in revised form, March 4, 1996)
From the
Anti-metatype (anti-Met) antibodies are immunoglobulins that specifically recognize and stabilize antibodies in their liganded or metatypic state, but lack specificity for either the hapten or the unliganded antibody. Autologous anti-Met antibodies were previously observed in vivo, suggesting that a metatypic autoantibody response could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from circulation. The first elicited anti-Met antibodies were against the fluorescein-liganded high affinity murine anti-fluorescein monoclonal antibody 4-4-20. The fluorescein-hapten system has proved to be an invaluable tool for both the recognition and characterization of the metatypic response by utilization of its spectral properties. In this investigation, hydrostatic pressure measurements, in conjunction with fluorescence spectroscopy, were performed on the recombinant Fv derivative (Fv 4-4-20) of the high affinity anti-fluorescein monoclonal antibody 4-4-20 complexed to anti-Met antibodies to study the influence of anti-Met antibodies on Fv 4-4-20 intervariable domain interactions. Anti-Met antibodies bound to liganded Fv 4-4-20 were observed to cause a change in the fluorescence properties of fluorescein that was not observed when anti-Met antibodies were bound to the liganded parent immunoglobulin. The variation of these spectral properties upon addition of anti-Met antibodies was shown to be correlated with dissociation of the variable domains in Fv 4-4-20 in response to its interaction with the anti-Met antibody. The ability to cause variable domain dissociation was dependent on whether monoclonal or polyclonal anti-Met antibodies were bound to the metatype. A model was proposed that elucidated the interaction of anti-Met antibodies, polyclonal and monoclonal, with variable domains of the primary anti-antigen antibody.
Anti-metatype (anti-Met) ()antibodies are
immunoglobulins that specifically recognize and stabilize antibodies in
their liganded state (or metatype antibodies), but lack specificity for
the hapten(1) . Autologous anti-Met antibodies were observed in vivo, suggesting that a metatypic autoantibody response
could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from
circulation(2) . The first anti-Met antibodies were elicited
against the liganded high affinity murine anti-fluorescein monoclonal
antibody 4-4-20(1, 3) . Since then, investigators have
elicited anti-Met antibodies against immunoglobulins specific for other
chemical compounds or peptides(4, 5, 6) .
These anti-Met reagents have demonstrated utility in the development of
improved analytical assays for small molecules and verified that
metatype
anti-Met complexes are not solely a property of
anti-fluorescein antibodies. However, detailed information about the
effect of anti-Met antibodies on the metatype, which would provide a
better understanding of the metatypic response, remains sparse.
The
fluorescein-hapten system has proved to be an invaluable tool for both
the recognition and characterization of the metatypic response by
utilization of its spectral properties(7) . Comparison of
solution- and solid-phase assays of mAb 4-4-20 and mAb
4-4-20anti-Met complexes (8, 9, 10, 11, 12, 13, 14, 15, 16, 17) has
yielded important information about the effect of the anti-Met
antibodies on the metatype. For example, dissociation rate analyses
demonstrated that the off-rate of the hapten from the metatype is
decreased in the metatype
anti-Met complex(18) .
D
O/fluorescein fluorescence enhancement studies showed that
delay in ligand dissociation involves a decrease in rate of
conformational fluctuations in the metatype antibody variable domains
when part of the metatype
anti-Met complex(19) . Solid-
and solution-phase studies of anti-Met antibody binding to the single
chain derivative scFv 4-4-20(20, 21) , a genetic
construct that links the variable domains of a corresponding monoclonal
antibody by a polypeptide linker(22, 23) , yielded
similar results. This indicated that metatopes (or anti-Met-binding
site) on the metatype that affected hapten dissociation are specific to
the variable domains(18) . In summary, it is clear that
anti-Met antibodies bound to a metatype variable domain have a
significant damping effect on metatype dynamics, which prolongs the
residence time of the hapten in the metatype active site.
An
assessment of the contributions of various metatype structural
components to the metatype/anti-Met interaction remains to be
addressed: 1) the role of constant domains of the metatype antibody and
2) the role of intervariable domain interactions. mAb
4-4-20anti-Met complexes were previously noted to have an
identical fluorescence intensity quenching maximum (Q
) and steady-state polarization (18) as the native liganded mAb 4-4-20. In contrast, scFv
4-4-20
anti-Met complexes showed decreased fluorescein
fluorescence Q
and polarization values (24) with regard to the parent immunoglobulin. This indicated
that anti-Met antibodies caused an additional disturbance at the
active-site level of the anti-hapten antibody in the scFv
4-4-20
anti-Met complex that did not occur in the mAb
4-4-20
anti-Met complex. A process that would have resulted in the
decrease of fluorescein Q
and polarization in
liganded scFv 4-4-20 was interdomain dissociation. Another process was
the dissociation of fluorescein from the metatype active site. In
total, these observations inferred that constant domains influenced the
steady-state metatype
anti-Met complex equilibrium by stabilizing
the metatype against the effects of binding an external reagent such as
the anti-Met antibodies.
To investigate this further, hydrostatic
pressure measurements, in conjunction with fluorescence spectroscopy,
were performed with the recombinant Fv derivative (Fv 4-4-20) (25) of the high affinity mAb 4-4-20 complexed to anti-Met
antibodies (Fv 4-4-20anti-Met complex) to study the effect of the
absence of metatype constant domain and intervariable domain
interactions on the stabilizing influence of anti-Met antibodies. Fv
4-4-20 was an ideal system for this study. Fv fragments are composed of
heavy (V
) and light chain (V
) variable domains
bound by noncovalent interactions, thus allowing investigation of
variable domain interactions in the absence of the constant domains of
mAb or the polypeptide linker of scFv. The recombinant Fv 4-4-20
derivative was recently constructed, expressed, and characterized using
solid- and solution-phase assays and was found to be identical to scFv
4-4-20 in all respects(25) . In this investigation, similar to
scFv 4-4-20(24) , Fv 4-4-20
anti-Met complexes showed
decreased fluorescein fluorescence Q
and
steady-state polarization compared with mAb 4-4-20. These spectral
properties were shown to be correlated with dissociation of the
variable domains of the metatype in response to interaction with the
anti-Met antibody. Although anti-Met antibodies caused a decrease in Fv
4-4-20 variable domain association, hydrostatic pressure measurements
demonstrated that when the hapten was locked into the Fv
4-4-20
anti-Met complex, it was stabilized against dissociation
relative to the native liganded Fv 4-4-20. Dissociation of the variable
domains observed with Fv 4-4-20 when bound by anti-Met antibodies did
not occur with immunoglobulins or Fab fragments because the constant
domains play a significant role in stabilizing intervariable domain
interactions. In vivo, dissociation of Fv domains by
autologous anti-Met antibodies may explain the enhanced degradation of
Fv compared with other immunoglobulin proteins stabilized by covalent
bonds(26, 27) .
where Q is the ratio of the fluorescence quantum yields
of free and bound forms of fluorescein, r is the anisotropy at
each pressure, and r and r
are anisotropies for free and bound states of fluorescein,
respectively, and are calculated from polarization as r = 2P/(3 - P), where P is
the polarization.
Hydrostatic pressure in the range of 1 bar to 2.4 kilobars was achieved using the pressure cell described by Paladini and Weber(33) . The sample was excited at 480 nm with a slit width of 8 nm. Emission spectra were recorded in the wavelength region spanning 500 to 600 nm with a slit width of 8 nm. Temperature was regulated with a circulating water bath. All stock solutions were prepared in 10 mM Tris-HCl, pH 8.0. The fluorescein fluorescence intensity from each protein sample was allowed to stabilize (4 min) after each pressure change before emission spectra were taken. Fluorescence intensity at each pressure was acquired by integrating the area under the emission spectrum.
Figure 1:
Anti-Met antibody titration curves for
liganded Fv 4-4-20 anisotropy in the presence of increasing
concentrations of monoclonal () and polyclonal (&cjs2110;)
anti-Met antibodies.
For the
Fv 4-4-20 affinity-labeled interdomain binding curves (see Fig. 2), fluorescein polarizations were obtained as a function
of affinity-labeled Fv 4-4-20 concentration at constant anti-Met
antibody concentration. Affinity-labeled protein was serially diluted
(1:2) over the concentration range of 23.5 µM to 4
nM. The domain dissociation was calculated using and was plotted as a function of Fv 4-4-20 protein
concentration. Fv 4-4-20 concentrations used in Fig. 2were
corrected for the R value or the amount of affinity-labeled
protein of Fv affinity labeling. The concentration of fluorescein was
determined on a Beckman DU-64 spectrophotometer using absorbance at 492
nm and an extinction coefficient () of 72,000
cm
M
. The R value or the amount of affinity-labeled protein was calculated as
the ratio of fluorescein to Fv 4-4-20 protein concentration. The
concentrations for the polyclonal and monoclonal 4A6 anti-Met
antibodies were constant at 200 and 118 µM, respectively.
Fluorescein was covalently coupled in the Fv 4-4-20 active site using
the isothiocyanate derivative (isomer I) of fluorescein (Sigma). A 1.1
molar excess of fluorescein isothiocyanate was incubated with Fv 4-4-20
for 4-5 h with agitation at 37 °C. Protein samples were
extensively dialyzed against phosphate-buffered saline to remove free
fluorescein isothiocyanate.
Figure 2:
Intervariable domain binding curves for
the affinity-labeled Fv 4-4-20 interdomain dissociation parameter
() as a function of affinity-labeled Fv 4-4-20 concentration in
the absence of anti-Met antibodies (
) and in the presence of
monoclonal (&cjs2110;) and polyclonal (
) anti-Met
antibodies.
In Fig. 2, affinity-labeled Fv 4-4-20 was used to monitor the effect of increasing anti-Met antibody concentrations on the intervariable domains by observing its effect on the Fv dimer dissociation concentration. Both monoclonal and polyclonal anti-Met antibodies caused a decrease in fluorescein anisotropy in affinity-labeled Fv 4-4-20 compared with that in the absence of anti-Met antibodies, indicating that domain-domain dissociation had occurred. As in Fig. 1, the monoclonal antibody had a greater effect on the fluorescein fluorescence anisotropy compared with the polyclonal antibody.
Figure 3:
Normalized fluorescein fluorescence
recovery for liganded Fv 4-4-20 at 0.075 µM () and
0.127 µM (&cjs2110;) Fv 4-4-20 active-site concentrations
and 0.02 µM fluorescein.
Pressure-induced dissociation of fluorescein from the active site of liganded scFv 4-4-20 had previously been confirmed by Coelho-Sampaio and Voss(14) . In that study, it was shown that when hydrostatic pressures were applied to liganded scFv 4-4-20, the increase in fluorescein fluorescence intensity below 1.6 kilobars was solely due to fluorescein dissociation. Fig. 4shows comparative pressure-induced dissociation curves for liganded Fv 4-4-20 and liganded scFv 4-4-20 at the same concentrations of active site and fluorescein. The identity of these two curves indicated that dissociation of fluorescein from the active site of Fv 4-4-20 was not due to the variable domain dissociation that would have been expected to occur in liganded Fv 4-4-20 because it was devoid of linker.
Figure 4:
Comparison of fluorescein fluorescence
recovery of liganded scFv 4-4-20 () and liganded Fv 4-4-20
(&cjs2110;) both at 0.127 µM active-site concentration and
0.02 µM fluorescein.
On the other hand, pressure-induced dissociation experiments performed on affinity-labeled Fv 4-4-20 as a function of protein concentration confirmed that the fluorescein fluorescence increase was due to a first-order reaction (data not shown). This was an expected result since the fluorescein covalently binds to the active site and cannot dissociate. Pressure was applied to affinity-labeled scFv 4-4-20 at an equal protein concentration to compare the role of the linker in pressure-induced fluorescein fluorescence recovery on affinity-labeled derivative antibodies. The intensity recovery for equal concentrations of affinity-labeled Fv 4-4-20 and affinity-labeled scFv 4-4-20 active sites demonstrated more intensity recovery for affinity-labeled Fv 4-4-20 (Fig. 5). The smaller fluorescence recovery for affinity-labeled scFv 4-4-20 compared with affinity-labeled Fv 4-4-20 indicated that the first-order reaction may be restricted by the linker. It was therefore concluded that the first-order reaction involved rearrangement of the fluorescein microenvironment, which was more restricted in affinity-labeled scFv 4-4-20.
Figure 5:
Comparison of fluorescein fluorescence
recovery for affinity-labeled scFv 4-4-20 (&cjs2110;) and
affinity-labeled Fv 4-4-20 () both at 0.150 µM active-site concentration.
Anti-Met antibodies were added to liganded Fv 4-4-20 to observe the effect on fluorescein dissociation as a function of pressure (Fig. 6). Comparison of the fluorescein dissociation of liganded Fv 4-4-20 with and without polyclonal anti-Met antibodies demonstrated that anti-Met antibodies hindered dissociation of fluorescein from the active site, as previously observed with liganded scFv 4-4-20 by Coelho-Sampaio and Voss(24) . Fig. 7shows the effect of pressure on fluorescein fluorescence enhancement of affinity-labeled Fv 4-4-20 and affinity-labeled scFv 4-4-20 with polyclonal anti-Met antibodies. Anti-Met antibodies stabilized affinity-labeled Fv 4-4-20 and affinity-labeled scFv 4-4-20 against fluorescein fluorescence enhancement in the two samples equally.
Figure 6:
Fluorescein fluorescence recovery of the
liganded Fv 4-4-20 complex in the absence (&cjs2110;) and presence
() of polyclonal anti-Met
antibodies.
Figure 7:
A, comparison of percent fluorescein
fluorescence recovery for affinity-labeled Fv 4-4-20 in the absence
(&cjs2110;) and presence () of polyclonal anti-Met antibodies. B, comparison of percent fluorescein fluorescence recovery for
affinity-labeled scFv 4-4-20 in the absence (
) and presence
(&cjs2110;) of polyclonal anti-Met
antibodies.
Fv 4-4-20, previously constructed and characterized by
Mallender et al.(25) , may be viewed as a protein
dimer. It exists as two pleated -sheet variable domains of
12-13 kDa lacking the interdomain polypeptide linker of scFv
4-4-20. The crystal structure of Fab 4-4-20 (35, 36) revealed a total buried surface area of 1366
Å
using van der Waals (119), hydrogen
bond(6) , and ion pair (1) interactions between the two
V
and V
domains for association. Hydrostatic
pressure had previously been shown to be a means of separating dimer
subunits without changing solvent parameters (33) or causing
substantial alterations of protein tertiary conformations at pressures
below 5 kilobars(34) . The results of Fig. 4are in
agreement with conclusions made by Mallender et al.(25) that Fv 4-4-20 antigen binding characteristics are
identical to those of scFv 4-4-20. At low concentrations, the linker in
scFv 4-4-20 minimizes dissociation of the variable domains compared
with Fv 4-4-20. However, at antibody concentrations above the K
, the interdomain interactions composed of the
previously mentioned van der Waals, hydrogen bond, and ionic pair
interactions appear sufficient to stabilize the domains against
interdomain dissociation as evidenced by the lack of discernible
difference between the derivative antibodies under application of
hydrostatic pressure.
The addition of polyclonal and monoclonal
anti-Met antibodies to liganded Fv 4-4-20 caused a decrease in
fluorescein fluorescence Q and polarization
similar to that previously observed by Coelho-Sampaio and Voss (24) with liganded scFv 4-4-20. In this study, the decrease in
liganded Fv 4-4-20 fluorescence Q
and
polarization when bound by anti-Met antibodies was examined more fully.
Coelho-Sampaio and Voss (24) attributed variation in
fluorescence Q
and polarization to idiotype
antibodies in the polyclonal anti-Met reagent. Anti-Met antibodies were
elicited against a specific primary liganded antibody by immunization
of a host with the affinity-labeled
antibodies(18, 19) . Anti-idiotype antibodies were
specific for unliganded antibodies and were generated in a similar
manner. Anti-idiotype antibodies present in the anti-Met reagent would
bind to the unliganded antibody, shifting the equilibrium between the
liganded and unliganded anti-fluorescein antibodies. The decrease in Q
would result from a greater amount of free
fluorescein than that present in the absence of anti-Met antibodies.
This explanation for the change in fluorescein fluorescence parameters
upon addition of anti-Met antibodies was not entirely satisfactory
because the same anti-Met reagent caused no appreciable effect on the Q
and
shift of liganded mAb
4-4-20. The absence of any variation in fluorescein fluorescence Q
and polarization upon addition of anti-Met
antibodies to liganded mAb 4-4-20 showed that the delay on the off-rate
by anti-Met antibodies was not a consequence of new bonds formed
between antigen and the active site. Conversely, the effect on the Q
and polarization of liganded scFv 4-4-20 bound
by anti-Met antibodies demonstrated that constant domains played an
important role.
To examine the mode of the effect of anti-Met on the
fluorescein fluorescence of liganded Fv 4-4-20, increasing
concentrations of anti-Met antibodies were added to liganded Fv 4-4-20 (Fig. 1). Monoclonal anti-Met antibodies, when present in large
excess, decreased the fluorescence polarization of liganded Fv 4-4-20
to a value that correlated with free fluorescein. Polyclonal anti-Met
antibodies decreased both Q and polarization,
but not as efficiently as monoclonal antibodies. The decrease in
polarization observed with the monoclonal antibody ensured that the
changes in Q
and polarization were not due to
anti-idiotype antibodies. To probe the effect of anti-Met antibodies on
the intervariable domain binding affinity, a constant concentration of
anti-Met antibodies was added to increasing amounts of affinity-labeled
Fv 4-4-20, and the fluorescein fluorescence anisotropy was plotted in
the form of a binding curve (Fig. 2). In affinity-labeled Fv
4-4-20, fluorescein was covalently bound to a lysine in the Fv 4-4-20
active site. A decrease in fluorescein fluorescence Q
or polarization upon addition of anti-Met was possible only if
the domains dissociated. At concentrations of Fv 4-4-20 below the K
, the interdomain dissociation was relatively the
same for affinity-labeled Fv 4-4-20, affinity-labeled Fv 4-4-20 with
monoclonal antibody 4A6, and affinity-labeled Fv 4-4-20 with polyclonal
anti-Met antibodies. At concentrations of affinity-labeled Fv 4-4-20
above the K
(25) , both monoclonal and
polyclonal anti-Met antibodies provoked domain dissociation. The
affinity-labeled interdomain binding curves (Fig. 2) indicated
that polyclonal and monoclonal anti-Met antibodies caused an
instability between the two variable domains that resulted in the
subsequent decrease in fluorescein polarization, which was in agreement
with Fig. 1. In conjunction, the results of Fig. 1and Fig. 2demonstrate that the effect of the monoclonal antibody on
the interdomain affinity was greater than that of the polyclonal
antibodies, and both provoked variable domain dissociation.
Hydrostatic pressure was used to examine the effect of anti-Met
antibodies on antibody active-site dynamics by using the
affinity-labeled antibody. Application of hydrostatic pressure in
affinity-labeled Fv 4-4-20 and affinity-labeled scFv 4-4-20
demonstrated an increase in fluorescein fluorescence intensity that was
the result of a first-order effect. The independence of fluorescein
fluorescence recovery as a function of protein concentration allowed a
comparison between affinity-labeled Fv 4-4-20 and affinity-labeled scFv
4-4-20 at the same protein concentrations. The greater increase in
fluorescence intensity observed in affinity-labeled Fv 4-4-20 compared
with affinity-labeled scFv 4-4-20 demonstrated that the first-order
reaction was restricted by the linker. Fluorophore fluorescence
intensity is known to be a sensitive measure of the fluorophore
microenvironment(7, 38) . The difference in the
results between affinity-labeled Fv 4-4-20 and affinity-labeled scFv
4-4-20 implied that the first-order reaction was most likely a
molecular rearrangement of the fluorescein active site. Fluorescein is
93% buried in the 4-4-20 active site across the interdomain
interface(38) . The difference in behavior between
affinity-labeled scFv 4-4-20 and affinity-labeled Fv 4-4-20 is probably
a reflection of the increased flexibility of the active site in Fv
4-4-20 due to the lack of a linker. Polyclonal anti-Met antibodies,
added to each of these affinity-labeled antibodies, were found to
stabilize against a pressure-induced fluorescein fluorescence increase.
This was interpreted as stabilization of the dynamic fluctuations of
the microenvironment of the fluorescein, and therefore the active site,
and was in agreement with DO enhancement studies (19) that showed anti-Met to stabilize conformational dynamics.
Pressure-induced ligand dissociation was performed on liganded Fv 4-4-20 to examine the efficiency of anti-Met antibody stabilization of fluorescein in an antibody active site devoid of interdomain polypeptide linker or constant domains (Fig. 6). Polyclonal anti-Met antibodies were found to stabilize fluorescein in the active site of liganded Fv 4-4-20 against dissociation with the same efficiency as for liganded scFv 4-4-20. The effect of polyclonal anti-Met antibodies on anti-fluorescein antibody ligand off-rate delay was antibody concentration-dependent, in agreement with results obtained with liganded scFv 4-4-20(24) . The concentration dependence was most likely because higher concentrations of anti-Met antibodies ensured that more Fv 4-4-20 variable domain metatopes were bound. In summary, polyclonal anti-Met antibodies had caused both the fluorescein release and stabilization in the Fv 4-4-20 active site. This apparent paradox is resolved in the following model proposed to explain general metatype/anti-Met interaction (Fig. 8).
Figure 8: Five-stage model outlining the dynamics of anti-Met antibody interaction with Fv variable domains. FL, fluorescein.
In
this model, a concentration-dependent five-stage equilibrium exists
between the individual variable domains (V and
V
), fluorescein, and anti-Met antibodies. Individual
domains are known not to bind fluorescein(25) . At variable
domain concentrations below the K
, the domains are
not associated with each other or with fluorescein (stage 1). At
concentrations above the K
, Fv dimer is formed
(stage 2). Upon formation of Fv dimer, fluorescein is quickly bound
(stage 3). Liganded Fv in the presence of anti-Met antibodies will form
a liganded Fv
anti-Met encounter complex (stage 4), which will
either go on to stage 5, the stabilized liganded Fv
anti-Met
complex, or back to stage 1, the basic components. The course of the
reaction from stage 4 to either stage 5 or 1 depends on the variety and
concentration of anti-Met antibodies.
Dimer destabilization observed
with monoclonal anti-Met antibodies is believed to be similar, but not
identical, to a phenomenon with the a-subunit of tryptophan
synthetase observed by Silva et al.(39) and bovine
lactate dehydrogenase by King and Weber(40) . Both groups found
that pressure-induced dissociation of the dimer caused the reassociated
species to have decreased subunit affinity. Bovine lactate
dehydrogenase showed reduced enzymatic activity upon removal of
pressure, although subunit reassociation occurred immediately. It was
concluded that upon dissociation of the aggregate, the subunits had
undergone a change in conformation that resulted in a loss in affinity
for each other(34) . Polyclonal anti-Met antibodies were shown
previously (31) and in this study with the pressure experiments
to have an effect on the antibody active-site dynamics. The
affinity-labeled anti-Met titration curve utilizing the monoclonal
anti-Met antibody, which by definition bound to one metatope on the
V
domain, showed the monoclonal antibody to have the effect
of destabilizing the dimer. Although only bound to one domain,
monoclonal anti-Met antibodies were previously shown to be capable of
stabilizing scFv 4-4-20 variable domain dynamics(30) .
Stabilization of one domain in the dimer by a monoclonal anti-Met
antibody would make the two domains dynamically incompatible, causing
dissociation to occur readily if no additional stabilizing element such
as constant domains or a linker is present. Therefore, for anti-Met
antibodies to stabilize ligand in an anti-antigen antibody-antigen
complex as observed in this and previous
studies(24, 30, 31) , the binding of an
anti-Met antibody to one domain on the metatype has to be balanced by
binding to the other domain. Indeed, this was observed when polyclonal
anti-Met antibodies were added to affinity-labeled and liganded Fv
4-4-20, where the decrease in Q
and polarization
was much less than for monoclonal antibodies ( Fig. 1and Fig. 2). Once the complex was formed, the metatype
anti-Met
complex was more resistant to hapten dissociation, as observed in Fig. 6and Fig. 7. In addition, this would explain the
lack of cooperativity observed by Coelho-Sampaio and Voss (24) when trying to stabilize liganded scFv 4-4-20 by using
different combinations of monoclonal anti-Met antibodies. All the
monoclonal anti-Met antibodies used in that investigation were specific
for the V
domain(30) . To observe cooperativity by
combining different monoclonal antibodies, an important requisite is
that monoclonal antibodies specific for both domains be combined. The
combination of monoclonal antibodies specific for both domains may
create the balance of the domain dynamics needed for metatype/anti-Met
stabilization. The small decrease in Q
and
polarization observed with liganded Fv 4-4-20 when bound by polyclonal
antibodies in Fig. 1was probably due to a population of Fv
dimers that were not bound on both domains.
Anti-Met antibodies were shown to affect the interdomain
interactions. The decrease in fluorescence Q and
polarization observed with affinity-labeled Fv 4-4-20 upon addition of
both polyclonal and monoclonal anti-Met antibodies indicated that some
incompatibility was initiated between the Fv 4-4-20 domains. When only
one domain was bound, as in the case of a monoclonal anti-Met
antibody(30) , the Fv interdomain associative interaction was
particularly weakened. Polyclonal anti-Met antibodies caused
fluorescein to dissociate from one fraction of the Fv 4-4-20 population
while further stabilizing fluorescein in the other fraction. The degree
of stabilization of the variable domains was believed to depend on
whether both variable domains were bound by anti-Met antibodies.
Monoclonal derivative antibodies have the potential to be used as agents for diagnosis and therapy of cancer(37) . Functional Fv fragments, which are smaller in mass and have reduced in vivo half-lives compared with immunoglobulins, have been found to be unsuitable for these purposes because of the in vivo dynamic dissociation of the dimer(26) . In accordance with the results in this study, the in vivo dynamic dissociation of the Fv dimer may be due to an autologous anti-Met antibody response elicited against Fv fragments that significantly contributes to Fv instability in vivo, favoring dimer dissociation.
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