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Immunology |
Departments of Nuclear Medicine [L. S. Z., E. Z. B., R. J. S., Q. H. Z., H. F. D.] and Cell Biology [M. D. S.], Albert Einstein College of Medicine, Bronx, New York 10461 [L. S. Z., E. Z. B., R. J. S., Q. H. Z., M. D. S.], and First University Teaching Hospital, West China University of Medical Sciences, Chengdu, 610041, P. R. China [H. F. D.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-azophenyl-arsonate as an antigen.
Isotype-matched monoclonal antibodies (mAbs) 36-65 and 36-71,
with identical epitope specificity but 200-fold differences in
affinity, were chosen as targeting agents. The relative in
vitro binding of 36-65 and 36-71 was compared with an
artificial "tumor" model in vivo using
antigen-substituted beads s.c. implanted into SCID mice.
Nonsubstituted BSA beads were implanted in the contralateral groin as a
nonspecific control. The efficacy of the targeting of
[125I]-labeled antibodies was assessed by the imaging of
animals on a gamma-scintillation camera using quantitative
region-of-interest image analysis over the course of 2 weeks and by
postmortem tissue counting. In vitro, both antibodies
bound well to the HD antigen, whereas only the high-affinity mAb 36-71
bound effectively to the LD antigen. In vivo,
high-affinity mAb 36-71 bound appreciably to both LD and HD beads. In
contrast, there was no specific localization of low-affinity mAb 36-65
to LD antigen beads, although the antibody did bind to the beads with
the HD antigen. Whereas the high-affinity mAb 36-71 increased its
binding to HD beads throughout the 14 days of observation, binding of
the high affinity antibody to LD beads and of the low affinity antibody
to HD beads plateaued between 10–14 days. These in
vitro and in vivo findings demonstrate that the
need for a high-affinity antibody is dependent on the density of the
target antigen. High-affinity antibodies bind effectively even with a
single antigen-Fab interaction, irrespective of the antigen
density. In contrast, low-affinity antibodies, because of weak
individual antigen-Fab interactions, require the avidity conferred by
divalent binding for effective attachment, which can only occur if
antigen density is above a certain threshold. An understanding of the
differential behavior of high- and low-affinity antibodies and the
impact of avidity is useful in predicting the binding of monovalent
antibody fragments and engineered antibody constructs and underlies the
trend toward development of multivalent immunological moieties.
Consideration of the relative density of the antigen on the tumor and
the background tissues may enable and even favor targeting with
low-affinity antibodies in selected situations. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various provocative mathematical models have been used to probe specific theoretical issues relating to antibody targeting, including the influence of affinity on binding (5, 6, 7, 8, 9, 10) . Fundamental behavior of antibodies has also been investigated in experimental in vitro studies (9, 10, 11) . Nonetheless, the extrapolation of these findings to clinical situations has proven uncertain (12, 13, 14) , and experimental validation in animal studies is needed. In fact, a limited number of in vivo studies have been performed to investigate factors influencing antibody binding (8 , 13 , 15, 16, 17, 18) , often with conflicting or unclear conclusions. It has proved difficult to identify antibodies with significantly different affinity but with the same class and subclass that bind to an identical epitope. Another frequent limitation of in vivo models is a restricted ability to modify relevant variables. For example, the influence of antigen density on antibody targeting has not been extensively studied in experimental models, largely because it is difficult to systematically vary this parameter in tumor xenografts. This is regrettable in that the density of an antigen determines the ability of a multivalent antibody to engage multiple antigenic sites simultaneously. In contrast with "affinity," which measures the binding of an individual Fab-binding site and antigen, the overall strength of interaction between a multivalent antibody and antigen is termed "avidity" (19) . This parameter is of paramount importance in predicting antibody binding in a given context (9, 10, 11) .
The goal of this paper is to investigate the relationship between antibody affinity, antigen density, and antibody targeting by applying a highly modifiable, well-controlled antibody system to in vitro and in vivo experimental models. For this purpose, we have chosen to study antibodies that bind to the well-characterized chemical hapten Ars on the basis of the spectrum of affinities available and the ability to modify the density of the antigen target. Investigations were initially performed in vitro using ELISA and bead-binding assays. This system was then extended into an in vivo targeting model using radiolabeled antibodies and an artificial "tumor" implanted s.c. in mice where variables relating to the immunoglobulin and antigen could be rigorously controlled. The efficacy of targeting was assessed by the serial imaging of live animals on a gamma-scintillation camera with quantitative image analysis and by postmortem tissue counting.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antigen.
Attachment of the hapten Ars to carrier BSA at specific
substitution ratios was performed as described previously
(22)
, and the resultant Ars-BSA was stored at -70°C
until use. For the purpose of the current studies, antigen with a ratio
of 1.8 Ars molecules per BSA molecule was selected for use as a HD
antigen, whereas antigen with a 0.15 Ars:BSA molar substitution ratio
was used as a LD antigen. Unconjugated BSA served as an
Ars-negative control target.
ELISA.
Interaction of antibody affinity and antigen density was illustrated by
ELISA based on the variation of a technique published previously
(22)
. Binding of the specific antibodies (mAbs 36-65 and
36-71) to HD and LD antigen plates was studied in duplicate. Unless
otherwise indicated, dilutions were performed in a solution of 1 mg of
BSA/100 ml of PBS, incubations were at 37°C, and volumes of 50
µl/well were used. Plates were washed three times between each
reagent (Microwash II; Skatron Instruments, Sterling VA) with PBS
containing 0.05% Tween 20.
Antigen was diluted in PBS to a concentration of 10 µg/ml and
distributed into disposable 96-well ELISA plates (Corning Glassworks,
Corning NY). Two h thereafter, plates were washed, blocked with a
solution of 1 mg BSA/100 ml of PBS (100 µl/well) and stored at 4°C
until used. At that time, plates were rewashed and Ars-binding
antibodies, at concentrations of 20 µg/ml, were serially diluted
(1:2) across the plate and incubated for 1 h. Alkaline-phosphatase
conjugated goat antimouse 
1 (Southern
Biotechnology Associates, Inc., Birmingham AL) at a concentration of 1
µg/ml was then added to the washed plates with a repeat washing
2 h later and the addition of substrate (-nitrophenyl
phosphate, disodium) dissolved in a bicarbonate buffer [0.001
M MgCl2/0.05 M
Na2CO3 (pH 9.8)].
Absorbance of the plates was read at 405 
, after color developed
(Multiskan MS; Labsystems).
Iodination.
For the in vitro and in vivo bead studies,
antibodies (mAbs 36-65, 36-71, and MOPC 21) were iodinated using the
Iodogen method (Pierce, Rockford Il; Ref. 23
) under mild
conditions with resultant specific activity of between 3–5 µCi/µg.
Typically, 150 µCi of [125I]NaI were
incubated with 24 µg of antibody in a volume of 100 µl for 40 min.
Free iodine was then separated from the iodinated proteins by
size-exclusion chromatography over a Sephadex G-25 column (Pharmacia,
Piscataway NJ). Typical reaction yields of 45–50% were obtained and
trichloroacetic acid precipitation revealed 93–98%
protein-bound activity.
Conjugation of Ars-BSA to Beads.
HD and LD Ars-BSA and control BSA lacking Ars were each conjugated to
commercially available, chemically activated beads (Reacti-Gel HW-65F;
Pierce, Rockford IL; nominal diameter of 32–63 µm) under identical
reaction conditions. Twenty-one mg of protein in 5 ml of 0.1
M borate buffer (pH 9.0) were added per 5 ml of beads.
After a 72-h incubation (4°C), beads were separated from solution and
1 M Tris buffer was added for 24 h to block remaining
active sites. Beads were then spun down and stored in PBS and 0.02%
azide at 4°C.
In Vitro Antibody Binding Assay.
Binding of the three radiolabeled antibodies (mAbs 36-65, 36-71, and
MOPC 21) to beads bearing HD, LD, and control antigen was studied using
duplicate samples per combination of antibody and antigen. Within the
upper chamber of a 0.45-µm cellulose acetate SPIN-X microtube
(Costar, Cambridge MA), 0.004 µg of radiolabeled antibody, 5 µl of
freshly washed antigen beads, and 200 µl of 1% BSA/PBS were combined
and agitated on a rotating platform at room temperature. Seventy-two h
later, supernatant was separated from the beads by spinning the
microtubes at 1500 rpm for 5 min, rinsing with 300 µl of 1% BSA/PBS,
and respinning. Duplicate supernatant and bead samples were counted on
a counter (Compugamma 1282; LKB Wallac, Turku, Finland), and the
mean fraction of bound antibody was calculated.
Murine Targeting Model.
SCID mice were chosen for these studies because they lack any
potentially confounding endogenous immune response (24)
.
Fifty µl of antigen-specific beads (HD or LD) were introduced into
the left inguinal region of anesthetized adult female mice by s.c.
injection, whereas 50 µl of antigen-negative control BSA beads
were introduced into the contralateral right side. Mice were prepared
at least 1 week before targeting studies to allow any acute changes
surrounding the beads to subside. Thyroid uptake of radioiodine was
blocked by the addition of three drops of Strong Iodine (Lugol’s)
Solution USP (Regional Service Center, Inc., Woburn, MA) per bottle of
drinking water from this time and throughout the duration of the study.
Two separate investigations were performed as described below. The first study characterized the time course of antibody binding and validated choice of an optimal time point for statistical comparison of the groups. The second investigation studied a large number of mice at a single time point after binding had peaked to generate statistically valid comparisons of antibody binding in the different groups.
Temporal Analysis of In Vivo Bead-Binding.
To determine the time course of antibody binding to antigen, the four
permutations of antigen density (HD and LD) and antibody affinity (mAbs
36-65 and 36-71) were studied in groups of four mice each. Mice were
injected by tail vein with 2 µg of
[125I]-labeled antibody. Over the course of 14
days, two mice per each group (one for the final image set) were
periodically anesthetized with i.m. Pentobarbital, and imaged for 5 min
in ventral projection using a dedicated animal gamma-camera imaging
system (Picker Dynacamera 4C; Picker International, Highland Heights,
OH; and NucLear MAC computer system, Scientific Imaging, Inc.,
Littleton, CO). The camera was peaked on 125I
using a 20% energy window, with collimation achieved by
perpendicularly placed radiography grids (Model 278032; Liebel
Flarsheim, Cincinnati, OH). Identically sized ROIs were placed over the
specific and control antigen beads, and a ROI-binding ratio was
generated on the basis of the ratio of left to right groin counts.
Values from duplicate animals were averaged for the initial six time
points whereas the solitary value was used for the seventh and final
point.
In addition, a single mouse per combination of antibody and antigen
density was sacrificed at each of four time points over the 14-day
period, and specific and control beads were dissected out of the
inguinal areas, weighed, and counted by -counter (Compugamma 1282;
LKB Wallac, Turku, Finland). A relative binding ratio was calculated
based on the ratio of counts bound per g of specific Ars-beads (left
groin) divided by those bound per g of nonspecific control beads (right
groin).
Statistical Comparison of In Vivo Bead-Binding.
The effect on the binding of antibody affinity and antigen density was
studied in a total of 28 animals consisting of two cohorts of mice,
which were pooled to increase sample size. Mice were prepared with
specific (HD or LD) and control beads as above, injected with one of
three radiolabeled antibodies (mAbs 36-65, 36-71, or MOPC 21), and
sacrificed after 10 or 13 days, after binding had generally plateaued.
Specific and control antigen beads were removed, weighed, and counted
by -counter as above. The percent injected dose/g of beads was
calculated as was the relative binding ratio between specific and
control beads. Relationships between the relative binding ratio,
antibody affinity, and antigen density were analyzed by two-way ANOVA.
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RESULTS |
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In Vitro ELISA.
Binding of mAbs 36-71 and 36-65 to HD and LD antigen is illustrated by
ELISA in Fig. 1
. Duplicate measurements
are shown and were highly consistent. On HD antigen, both antibodies
bound effectively, with the higher-affinity mAb 36-71 binding to a
quantitatively greater degree than mAb 36-65. On LD antigen, a marked
qualitative difference in binding between the two antibodies was noted.
mAb 36-71 bound effectively to the LD antigen plate, whereas, for all
intents and purposes, the low-affinity mAb 36-65 did not bind. The
necessity of a HD target for effective binding of a low-affinity
antibody reflects the fact that individual antigen-Fab interactions are
weak and highly reversible and require the divalent attachment of both
Fabs from each IgG molecule. This higher-avidity interaction can occur
only if antigen density is above a certain threshold. High-affinity
antibodies, in contrast, are able to bind effectively even with a
single antigen-Fab interaction, and therefore are retained well on both
HD and LD targets.
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. Duplicate measurements
were all within 2.5%. The slightly higher binding of mAbs 36-65 and
36-71 to control BSA beads as compared with the MOPC 21 isotype-matched
control antibody was a consistent finding, suggesting weak
cross-reactivity of the specific antibodies for the BSA-beads.
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In Vivo Targeting.
The ultimate goal of our study was to examine antibody behavior
in vivo. The time course of binding was demonstrated over a
14-day period in adult female SCID mice. Fifty µl of antigen-specific
beads (HD or LD) were introduced into the left inguinal region by s.c.
injection, whereas 50 µl of antigen-negative BSA beads were similarly
introduced on the right side. One week later, mice were injected with 2
µg of 125I-labeled mAbs 36-65 or 36-71. Two
animals per group (one in the final session) were periodically
anesthetized and imaged (Fig. 2)
.
Identically sized ROIs were placed over the specific and control
antigen beads, and a ROI binding ratio was generated based on an
average value per time point (Fig. 3)
. On
sequential gamma-camera images (Fig. 2)
, activity is initially noted in
the vascular structures, with prominence of the heart, great vessels,
and viscera, especially the liver. Over time, this activity lessens as
the blood pool activity decreases. It is interesting to note a mild
amount of activity which was localized to the control beads
(right groin) in all groups of animals. This nonspecific accumulation
may be attributable in part to increased vascular and interstitial
volume surrounding the beads as well as to a mild degree of
cross-reactivity of the antibodies with control BSA beads, as noted in
the in vitro bead study (Table 1)
. The presence of
nonspecific uptake highlights the need to compare specific localization
of antibody to antigen-positive beads in the left groin with the mild
degree of nonspecific localization in the right groin. No specific
localization of low-affinity antibody to LD antigen was noted
(column 1), although specific binding was apparent on HD
antigen (column 3). High-affinity mAb 36-71 bound
appreciably to both LD antigen (column 2) and especially to
HD antigen (column 4). Visual impression was quantitated and
validated by the ROI analysis (Fig. 3A)
.
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). Binding of
low-affinity mAb 36-65 to the LD antigen was similar to control
antigen, with a ratio of 1, whereas this antibody did bind effectively
to the HD antigen target. High-affinity mAb 36-71 bound specifically to
both the LD and especially to the HD antigen. As a general rule,
antibody binding appeared to plateau between 10–14 days, validating
the choice of this interval for use in the quantitative analysis
described below. mAb 36-71 binding to HD beads was not only the highest
for each observation, but continued to increase throughout the duration
of the study. This is not surprising, in that the high affinity and
avidity of interaction between 36-71 and the beads would dictate that
any available antibody would continue to bind to the beads, whereas
effectively none would be lost by elution. Visual (Fig. 2)
and ROI
observations (Fig. 3A)
were consistent with the quantitative
postmortem bead measurements (Fig. 3B)
, though somewhat
blunted in magnitude. Decreased ratios for the imaging data are not
surprising based on the degrading effects of overlapping background
tissues, limited camera spatial resolution, and the attenuation by
overlying soft tissues. These technical factors may explain the
inability to obtain correlation between absolute counts and imaging
results in a previous study by Andrew et al.
(16)
.
Binding of control, low-, and high-affinity antibodies was also
compared in a larger group of 28 mice to enable valid statistical
analysis. SCID mice bearing HD or LD antigen, prepared as above, were
injected with 2 µg of 125I-labeled mAbs 36-65,
36-71, or MOPC 21. After 10 or 13 days, mice were sacrificed and
specific and control antigen beads were dissected, weighed, and
counted. The absolute binding of antibody to the antigen and control
beads was expressed as a percentage of injected dose/g of beads
(%ID/g; Table 2
). A relative binding
ratio was also calculated based on the ratio of counts bound/g of
specific beads (left groin) divided by those bound to the nonspecific
control beads (right groin).
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. Binding of MOPC 21
control antibody to the HD or the LD antigen target did not differ
appreciably from binding to contralateral control BSA beads, with
localization ratios statistically indistinguishable from 1
(P = 0.25 and 0.50, respectively). Binding of
low-affinity antibody to the LD antigen was also indistinguishable from
the control groups and was statistically similar to 1, indicating a
lack of effective targeting (all Ps >0.85). Binding of the
high-affinity mAb 36-71 to the HD antigen was statistically superior to
all other groups (P = 0.0001) with a
localization ratio of 10.5 ± 3.0. Binding of
high-affinity mAb 36-71 to LD antigen beads was intermediate in
strength and similar to the binding of the low-affinity antibody on HD
beads (P = 0.80), although different from
each of the other four groups (Ps <0.05). The qualitative
change in the binding of mAb 36-65 when binding LD versus HD
antigen, as contrasted with the relatively similar behavior of mAb
36-71, indicates a statistically significant interaction between
antibody type and antigen density. |
DISCUSSION |
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It is interesting to consider how the present findings can be generalized to actual clinical tumor targeting. In the present paper, a difference in the ability to bind divalently was noted, although the HD and LD antigens only varied in density 12-fold. These concentrations were, in fact, chosen to demonstrate a dramatic difference in divalent binding, even within a small range of densities, without confounding secondary factors such as the total number of binding sites or mass-action. In more extreme examples of HD and LD antigen, antibody would still be only able to bind to LD antigen monovalently, whereas divalent binding could occur with HD antigen. In vivo, it is difficult to theorize what concentration of antigen would enable divalent binding. One could compare the radius of the binding arms of an IgG molecule with the average density of antigen on the cell surface, but this would ignore the ability of antigen to migrate in the fluid cell membrane. Many antigens, such as mucins, also contain repetitive epitopes, which would have to be analyzed. Furthermore, issues such as accessibility of antigen, steric hindrance, and the flexibility of the IgG molecule would be difficult to predict. Perhaps the best proof regarding applicability of these concepts to actual tumor targeting is the fact that a large array of multivalent constructs, discussed below, has been shown to be qualitatively superior to univalent molecules in clinical and experimental studies.
In fact, the present findings provide interesting insights into clinical studies and suggest potential novel strategies for improving target-to-background localization. Clearly, the critical parameter for predicting antibody binding is avidity rather than affinity, because it takes into account the effect of multivalent interaction. A decrease in binding caused by reduced valence has been shown experimentally (25) and has long been clinically recognized when targeting enzymatically generated Fab and Fab' antibody fragments. This observation has taken on new relevance as novel immunological molecules are designed using recombinant DNA techniques. Many of the initial constructs developed, such as scFvs (26 , 27) were monovalent, and consequently exhibited relatively poor binding. To compensate, divalent constructs were devised, such as the divalently linked scFvs (28) , diabodies (29) , and minibodies (30) . In carefully controlled animal studies, the advantages of divalent constructs have been shown to be independent of the size and rate of vascular clearance (28 , 31) . Beyond producing small divalent constructs, other efforts have been expended to artificially increase the valence of immunologically derived molecules to supranormal values such as trimers or tetramers of Fab fragments (32, 33, 34) or multimers of intact IgG molecules (35, 36, 37) . This trend, in fact, recalls an earlier observation by one of the current authors (M. D. S.) regarding mutant antibodies spontaneously derived from the 36-65 cell line which demonstrated increased antigen binding to low-density Ars-BSA. Analysis showed that the mutations were not in the variable region increasing affinity, as might have been expected, but were, rather, in the constant region, leading to polymerization (22 , 38) . The consequent increase in span of the immunogobulin and valence resulted in >100-fold increases in antigen binding, as a manifestation of increased avidity.
The benefit of increasing the valence of the binding molecule would only be effective in the presence of antigen density adequate for multivalent binding. In clinical practice, a low-affinity divalent antibody may be quite adequate for targeting a specific tumor expressing a high density of antigen on its surface, but totally inadequate for a different tumor with a lower density of antigen. Experimental strategies to improve antibody targeting by increasing antigen density, such as biological response modifiers (reviewed in Refs. 39 and 40 ), can therefore be understood not only quantitatively as a means of increasing the total number of antigen-binding sites, but also qualitatively, as a strategy to increase the avidity of binding, which is especially important when antibody affinity and baseline antigen density are low. The importance of total antigen in driving the kinetics of antibody-antigen interaction has been considered previously in theoretical discussions (8 , 41) and in animal experiments (42) , although the qualitative effect of antigen density on avidity has not been singled out in these discussions.
It is interesting to contemplate tailoring the antibody affinity and valence to the specific clinical usage at hand. Taking into account the relative density of antigen on target and nontarget tissues, it could be possible to select antibodies of specific affinity and valence so as to better differentiate tissues bearing HD and LD antigen. For example, if an epitope is present at a high density on the tumor target and more sparsely on benign tissues, then it would be advantageous to choose a lower-affinity multivalent antibody so that avidity can be used to discriminate between binding on the two tissues. In an analogous manner, avidity may be the key to understanding the initially surprising absence of interference by some circulating antigens when targeting tumors with radiolabeled antibodies. Because interaction between an antibody and many soluble antigens is often monovalent, overall avidity of binding is low. This allows antibodies of low-to-moderate affinity to readily dissociate from circulating antigen and to bind in a more irreversible manner to the target tumor where antigen density, and hence avidity, is greater (22) . This difference in binding would be lessened in the case of high-affinity antibodies, which could presumably bind irreversibly to circulating antigen even monovalently, or when targeting with monovalent fragments, which do not bind multivalently to the tumor antigen. In these cases, circulating antigen may conceivably represent a greater problem for tumor targeting.
When targeting with monovalent antibody fragments, the present observations suggest that binding would succeed best with higher affinity antibodies. This is in accordance with the observations of Adams et al. (43) , who generated a set of scFv molecules with increased affinity by site-directed mutagenesis. Using this well-controlled family of reagents to target a SCID mouse xenograft model, increased affinity resulted in improved tumor targeting. In his model system, Adams has also noted that, above a given threshold, no additional benefit is realized (44) . The existence of a ceiling of affinity above which no additional advantage is obtained should be even more evident when targeting with multivalent immunological molecules. Whereas a small experimental literature comparing divalent antibodies of differing affinity has appeared (13, 14, 15, 16, 17, 18 , 45, 46, 47) , it is difficult to extrapolate from many of these studies because of unclear results or the presence of confounding factors, such as differences in the epitope targeted.
Much has also been made of the provocative, yet persuasive, arguments of Weinstein et al. (5) and Fujimori et al. (6) , who discussed the effect of elevated affinity on homogeneity as well as on total uptake by tumor nodules. Their modeling work suggested that an overly high affinity might be detrimental in therapy applications because the antibody would tend to accumulate at the periphery of tumor deposits. This phenomenon, which they termed the "binding site barrier," would thereby deprive the innermost tumor of therapeutic effect. In fact, this concept has been supported by experimental data (48) and certainly would be relevant in situations of elevated avidity attributable to multivalent binding.
Conclusion.
We have investigated the relationship between antibody affinity,
antigen density, and antibody targeting using modifiable,
well-controlled in vitro and in vivo experimental
models based on the chemical hapten Ars and isotype and epitope-matched
monoclonal antibodies that vary 200-fold in affinity. In contrast to
high-affinity antibody, which binds effectively to both HD and LD
antigen, low-affinity antibody only binds appreciably to HD antigen
because of its requirement for divalent attachment. In the rational
design of immunological reagents for specific in vivo
applications, the impact of antibody affinity must be viewed in the
context of the antigen density of the target and background tissues.
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FOOTNOTES |
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1 L. S. Z. was partially supported by NCI 1K11
CA01503, and M. D. S. was supported by R35CA39838, R01CA72649, and
the Harry Eagle Chair from the National Women’s Division of the Albert
Einstein College of Medicine. 
2 To whom requests for reprints should be
addressed, at Department of Nuclear Medicine, Albert Einstein College
of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461.
3 The abbreviations used are: mAb, monoclonal
antibody; Ars, -azophenyl-arsonate; HD, high-density; LD,
low-density; ELISA, enzyme-linked immunosorbent assay; ROI, region of
interest; SCID, severe combined immunodeficiency; scFv, single-chain
variable region.
Received 5/18/00. Accepted 9/ 1/00.
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