From the Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle,
Washington 98109-1024
The crystal structure of the complex between hen egg lysozyme and the Fv fragment of a humanized antilysozyme antibody was determined to 2.7-Å resolution. The structure of the antigen combining site in the complex is nearly identical to that of the complexed form of the parent mouse antibody, D1.3. In contrast, the combining sites of the unliganded mouse and
humanized antilysozymes show moderate conformational differences. This disparity suggests
that a conformational readjustment process linked to antigen binding reverses adverse conformations in the complementarity determining regions that had been introduced by engineering
these segments next to human framework regions in the humanized antibody.
 |
Introduction |
Humanized antibodies were designed to limit the response of the human immune system to rodent monoclonal antibodies used in therapy of human disease (1). They
represented an advance over chimeric antibodies (2), which
were engineered with murine immunoglobulin variable domains and human constant domains, in that the foreign
content of humanized variable regions was substantially reduced. This was achieved by combining the short, hypervariable complementarity determining regions (CDRs)1 of
a murine antibody, which fold to form an antigen combining site of unique structure, with human variable domain
framework regions, which appear to be conserved in sequence throughout all races (5). The resulting molecule has
the same specificity as the murine antibody, but substitution of human sequences confers a much longer in vivo
lifetime and nearly eliminates immunogenic side effects
(10).
Framework and CDR segments can be identified from sequence information alone, since they are defined by homology rather than structure (11, 12). At the three-dimensional
level, however, the two sets of residues are in intimate contact and mutually influence each other's conformation (13,
14). Human framework residues can alter the conformation
of transposed mouse CDRs and thereby disrupt antigen binding. Since human and mouse framework regions differ by
upwards of 50 out of 170 residues (15), the potential for
this sort of disruption is high. Nevertheless, humanizing by
grafting mouse CDRs onto human frameworks usually
transfers antigenic specificity, though sometimes additional
framework mutations are required to put antigen affinity
on a par with the starting mouse antibody (13).
In this report, we describe intrinsic aspects of immunoglobulin structure that may partly account for the ease with
which CDRs and framework regions from different animal
species and unrelated antigenic specificities may be combined to give a functional molecule. We have determined
the crystal structure of the complex between hen egg white
lysozyme and the immunoglobulin heavy and light chain
variable domains (Fv) of a humanized version of the mouse antilysozyme D1.3 (HuLys). This is the first reported structure of a complex of antigen with a humanized antibody, and
completes a family of crystallographically determined component structures. D1.3 was previously determined in the
free form and complexed with lysozyme, both at 1.8-Å resolution (16, 17). HuLys was determined in the unliganded
form at 2.9-Å resolution (14) . The human immunoglobulins NEW and REI, from which the HuLys heavy and light
chain framework regions were taken, respectively, have previously been determined in their unliganded form, both
at 2.0-Å resolution (18). The availability of mouse, human, and antigen complex structures for reference has now
allowed us to identify conformational differences in the humanized Fv that result from protein engineering and ligand
binding.
 |
Materials and Methods |
Protein Expression.
The HuLys protein used in this study was
a high affinity form with variable domain sequences identical to
those published previously (21). This molecule was expressed as
an Fv fragment in the Escherichia coli strain 25F2, using the vector
pAK19 (22). In brief, 15 liters of defined medium (23) supplemented with 0.15% casamino acids, 1% glucose, and 20 µg/ml
tetracycline was inoculated with 400 ml of a starter culture (absorbance at 600 nm 1.5-2) and grown in a fermentor (New
Brunswick Scientific MPP, Edison, NJ) for 20 h at 37°C. Cells
were harvested by centrifugation and subjected to osmotic shock
to release periplasmic proteins (24). The periplasmic fraction was
clarified by centrifugation (30 min at 14,500 g) and passed
through a lysozyme-Sepharose column. After extensive washes
with PBS and high salt buffer (500 mM NaCl, 50 mM Tris, pH
8.5), the Fv was eluted with 50 mM diethylamine.
Crystallization.
The purified Fv was complexed with lysozyme
and crystallized from phosphate buffer. Lysozyme (three times
crystallized) was purchased from Sigma Chemical Co. (St. Louis,
MO) and dissolved in PBS. HuLys-lysozyme complex was prepared by mixing the two in equimolar proportions, letting the solution sit for 30 min, and diluting to 10 µg/ml with PBS. There
was immediate slight cloudiness upon mixing; the solution was
spun in a bench-top centrifuge before setting up crystallizations.
Crystallization was by vapor diffusion; both hanging drops and
sitting drops in microbridges were used. Protein concentration
was 7 or 10 mg/ml. The reservoir was 1.6 or 1.7 M phosphate
(made by mixing equal volumes of K2HPO4 and NaH2PO4), and
0.1 M Hepes, pH 6.5. Equal volumes of protein and reservoir solutions were mixed to make the drop.
Data Collection.
Two x-ray diffraction data sets were collected to
2.9-Å resolution and 2.7-Å resolution, each from a single crystal
at 4°C, using an r-axis detector. The two data sets were processed
(Table 1) with DENZO and SCALEPACK (25). The lower resolution data set, truncated to 3.5 Å, was used initially for determining the molecular replacement solution. Refinement was carried
out when the higher resolution data set became available.
Molecular Replacement Solution.
A search model for molecular
replacement was constructed by superposing molecule 2 of uncomplexed HuLys (14), the molecule whose CDRs are less affected by crystal packing, upon the murine complexed structure
and combining the HuLys and lysozyme structures. Molecular replacement was carried out using the program AMoRe (26) with
the 3.5-Å resolution data set. The space group was determined to
be P41212 from the four possibilities P4x212, x = 0, 1, 2, 3, by solution of the translation function. (There were no 00l reflections
in the data set.) The unit cell dimensions were a = b = 97.7 Å,
and c = 174.9 Å. After refinement of the translation functions for
the two molecules in the asymmetric unit, the correlation coefficient was 0.63 and the R value was 0.38 (15-3.5 Å).
Crystallographic Refinement.
The 2.7-Å resolution data set was
partitioned by X-PLOR (27) into two sets, one for refinement
and calculation of the working R value, and the other for calculation of the free R value (28, 29). A rigid-body refinement was
performed at 3.5-Å resolution using X-PLOR, first with each
complex as a rigid body, and then with each chain as a rigid body.
The refinement resulted in a drop of both working R and free R
from 0.46 to 0.32 (10-3.5 Å). Alternating rounds of positional
and individual B value refinement, using both X-PLOR and
TNT (30), and model building, using QUANTA (Molecular
Simulations, Inc., Burlington, MA), were performed (Table 2).
No solvent molecules were included in the model. The values for
working R and free R dropped from 0.35 to 0.21 and from 0.34 to 0.30, respectively (10-2.7 Å). A PROCHECK analysis (31) of
the structure showed no residues in disallowed regions of a Ramachandran plot except for residue L51 of both molecules. This is
also seen in the uncomplexed HuLys and REI and many other Fab structures. The residue is in a
-turn conformation (32). Residue numbering follows the Kabat system (15). In this paper we
precede each residue number with a chain designator, e.g., L51
for light chain residue 51.
 |
Results |
Antigen-Antibody Interaction.
The most striking feature
of the structure is apparent when the antigen-contacting
residues in the mouse and humanized Fv-lysozyme complexes are compared to the same residues seen in the free
Fv structures. The unliganded structures are shown superposed in Fig. 1 A. A multitude of conformational differences are observed between the unliganded mouse and
humanized combining sites. In contrast, the antigen-contacting residues in the HuLys and D1.3 complexes have
virtually identical conformations (Fig. 1 B). Although these
superpositions were calculated using only alpha carbon
(C
) atoms of the polypeptide chains, it is clear that the
side chain conformations are also nearly identical in the
complexed structures.

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Fig. 1.
Antigen combining site of
free and complexed HuLys and D1.3,
wall-eyed stereo views. (A), unliganded
forms. Molecule 2 of free HuLys (14) is
shown superposed on free D1.3 Fv. (B),
Complexed forms. Molecule 1 of the HuLys-lysozyme complex crystal form is shown superposed on D1.3 Fv from the
D1.3-hen lysozyme complex. Lysozyme
has been removed from the illustration.
Superpositions were performed with
QUANTA, using the C atoms of antigen-contacting residues. A list of
lysozyme-contacting residues in D1.3,
which match those seen in the HuLys-
lysozyme complex, is given in reference 35. The structures are shown from analogous "antigen eye views" looking directly
at the antigen combining site of each antibody. For simplicity, residues that do not
directly contact antigen are drawn as a C
trace, whereas antigen-contacting residues are fully drawn. HuLys, red; D1.3,
purple. The illustration was drawn using
the program MOLSCRIPT (46).
|
|
Several of the differences between complexed and uncomplexed forms in Fig. 1 occur not because of antigen
binding, but because the antigen-binding residues in the
uncomplexed Fv are involved in crystal packing interactions. The largest shift amongst antigen-contacting residues
of the murine Fv (0.69-1.06 Å) occurs for heavy chain residues H96-H98 (16), and probably arises from contacts
with symmetry-related molecules in the uncomplexed Fv.
A comparison of the uncomplexed and complexed forms
of HuLys Fv shows that the largest shift amongst antigencontacting residues occurs for residue L93; the C
-C
distances after CDR superposition are 1.20 and 1.31 Å for
molecules 1 and 2, respectively, versus molecule 2 of the
uncomplexed Fv structure. This large shift is not seen upon
examination of the D1.3 structures, possibly because in uncomplexed D1.3, this residue may be held in a complexed-like conformation due to crystal packing interactions. All
other shifts in antigen-contacting residues are less than
twice the root mean square (rms) distance for the superposition of all six CDRs.
The rearrangements accompanying lysozyme binding
represent a broader effect than symmetry-related intermolecular contacts alone can account for. Quantitative evidence that the CDRs of the liganded forms of HuLys and
D1.3 are more structurally similar than in the free Fv molecules is presented in Table 3. The rms differences in position of C
atoms in the mouse and humanized CDRs,
which comprise 56 residues in each molecule, is 0.37 Å when the Fv-lysozyme complexes are compared, versus
0.63 Å comparing the CDRs in the free Fvs (33). The
liganded CDRs are closer in structure than any other residue subset in Table 1. In addition, the binding of lysozyme
by HuLys causes the CDRs to more closely resemble the
unbound D1.3 CDRs. Even superposition of the lysozyme molecule in the HuLys complex onto the lysozyme molecule in the D1.3 complex gives a rms distance one fourth
larger than for the corresponding CDRs. Lysozyme itself
forms crystal packing interactions that differ in the mouse
and HuLys complexes, which give rise to some of the differences.
Contacts between HuLys and lysozyme were identified
using the program PAIRS (34). An analysis of these contacts is given in Table 4. Only direct protein-protein hydrogen bonds are considered, as we have not included water
molecules in the HuLys structure due to its lower resolution. Solvent molecules do in fact contribute greatly to the
stabilization of the complex, as was seen in the higher resolution D1.3 complex structure (35). The hydrogen bond
distances in the D1.3 and HuLys complexes are similar,
with those in HuLys marginally longer. Hydrogen bonds
between HuLys and lysozyme seen in both HuLys complexes are seen in all cases as well in the D1.3-lysozyme
complex. This comparison of antibody-antigen contacts in
the HuLys and D1.3 complexes was extended to include van der Waals interactions. The comparison showed three
interactions in D1.3 closer than 4 Å, between Trp H52 and
lysozyme Gly 117, which are not seen in HuLys. No interactions were found in both HuLys molecules that were not
also seen in D1.3.
We interpret the shift of the HuLys antigen contacting
residues from rough congruence to exact congruence with
the cognate residues in D1.3 as evidence for a conformational correction mechanism that allows the antibody to attain precise stereochemical complementarity with antigen.
The unliganded conformation of HuLys observed crystallographically (14) is unlikely to interact with the antigen as
strongly as the conformation both HuLys and D1.3 adopt
in the complex. During the antigen recognition process,
the HuLys combining site must undergo a correction to the
more favorable lysozyme binding conformation. Exactly
such a mechanism was hypothesized in the initial report of
humanizing the D1.3 heavy chain (36). X-ray data do not
allow us to distinguish whether this involves an "induced
fit" rearrangement of combining site residues after initial
complex formation (37, 38) or isomerization of HuLys to
the high affinity form before antigen encounter (39).
D1.3 itself undergoes a small conformational change accompanying lysozyme complex formation (16), but this differs somewhat from the change in HuLys, as evidenced
by the moderate disparity in D1.3 and HuLys unliganded
conformations.
Conformational Correction in Framework.
A second conformational correction mechanism appears to involve a
subtle rearrangement of HuLys framework residues proximal to the CDRs. Evidence for this rearrangement is
shown in Fig. 2. In 2 A, the framework of the Fv structure
has been divided into layers according to CDR proximity.
Residues within 4 Å of a CDR form the first layer, residues
approaching between 4 and 8 Å the next layer, and so on.
Fig. 2 C shows, for each layer, the degree of sequence
identity between HuLys and cognate residues in the two
parent antibody molecules. Here, HuLys and D1.3 have 100% sequence identity in the CDRs, whereas in all framework layers HuLys and the human molecules are more
similar in primary structure. Fig. 2 B depicts a parameter
more relevant to homology at the three-dimensional level.
Here, the distance from each HuLys C
to the cognate C
in superposed D1.3, REI, or NEW molecules was determined. The difference between the HuLys to human and
HuLys to mouse distance was then calculated, and the median for each residue layer was compiled. This difference is
a measure of whether a particular layer of HuLys is generally more similar in conformation to D1.3 or to REI and
NEW. We found that the CDRs of the mouse and humanized molecules are much more structurally similar than the HuLys CDRs are to the CDRs of NEW and REI.
Likewise, in the distal layers of the Fv, the humanized
framework is very similar to the human molecules at the
three-dimensional level. Although the sequence-structure
correlations at opposite poles of the Fv are intuitive and
unremarkable, the sequence-structure correlation in layers
of framework residues near the framework-CDR interface is counterintuitive. The humanized sequence remains very
close to the human sequences, but the framework of the
humanized molecule is conformationally more similar to
the mouse structure. The HuLys CDRs, which have virtually no homology to NEW and REI, thus appear to induce
a D1.3-like conformation in adjacent framework layers.

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Fig. 2.
CDR-induced changes in framework conformation. (A) C
trace of the HuLys Fv from molecule 1 of the HuLys-lysozyme complex crystal form, stereo view. Framework layers are colored according to
CDR proximity. CDRs are colored red. Framework residues that have at
least one nonhydrogen atom within 4 Å of any nonhydrogen CDR atom
are colored maroon. Residues whose nearest atom to a CDR atom is
within 4-8 Å are colored purple, within 8-12 Å colored green, and 12-27
Å colored black. (B) Conformational similarity between HuLys and D1.3
or HuLys and REI/NEW, by framework layer. Heavy and light chains of
the mouse and human structures were independently superposed on HuLys molecules 1 and 2 using PDBFIT (33). The distance between cognate
C atoms was calculated, and for each cognate set the value of the HuLys
to D1.3 distance was subtracted from the HuLys to REI/NEW distance.
The median difference for each framework layer is shown in the bar
graph. A positive value indicates that the HuLys to human distance is greater than the HuLys to mouse distance for a majority of residues in that
layer, and therefore that the HuLys structure is less similar to the human
and more similar to the mouse structure for that layer. For these calculations we used a total of 112 CDR C atoms in the two crystallographically independent HuLys Fv molecules, 92 in the 0-4 Å layer, 88 in the
4-8 Å layer, 74 in the 8-12 Å layer, and 80 in the 12-27 Å layer. (C) Sequence identity between HuLys, D1.3, and REI/NEW, by framework
layer. Open bars, HuLys compared to REI or NEW; filled bars, HuLys
compared to D1.3. The humanized, mouse, and human protein sequences were aligned by Kabat residue number. The number of positions
with identical residues in HuLys and D1.3 or HuLys and REI/NEW
were then tabulated for each framework layer, and this value is presented
in the figure as a percentage. The illustration was drawn using the program MOLSCRIPT (27).
|
|
 |
Discussion |
In HuLys, and possibly many other humanized antibodies, a heirarchy of immunoglobulin structural properties allows humanizing to "work". As a first approximation,
mouse CDRs retain function on human frameworks because backbone conformations of hypervariable loops usually follow one of a small number of canonical structures regardless of chemical environment (42). Human frameworks can usually be found that will support a set of mouse
CDRs identically to the mouse frameworks with which the
CDRs were first isolated. In HuLys, we have identified a
fine-tuning mechanism in which the mouse CDRs force
nearby humanized framework residues toward the backbone conformation found in the corresponding D1.3 frameworks. The implication for humanizing is that although point
mutations may have to be introduced into the frameworks
to improve antigen binding by a humanized antibody, generally only a few are required, and these are necessary to recreate
local structural motifs, such as the CDR-separating side chain
at H71 (43). Finer-scale tailoring of framework structures
appears unnecessary. At the combining site, the conformation of a humanized antibody free in solution may differ
somewhat from that of the parent mouse antibody, as was
found by comparing HuLys and D1.3 (14). However, a second conformational correction mechanism compensates for
imprecisions in lock and key complementarity that would
otherwise result from apposing rigid bodies. Interaction
with antigen erases these differences by selecting or inducing a conformation of HuLys nearly identical to that in the
D1.3-lysozyme complex. A concomitant shift towards the
uncomplexed mouse antibody conformation occurs as well.
Lastly, 25 tightly ordered water molecules form a layer at
the D1.3-lysozyme interface (35, 44, 45). These water
molecules bridge many antibody-antigen contacts, are thermodynamically significant in the antigen-binding reaction,
and many are conserved in free, complexed, and mutant
D1.3 structures, and hence must be considered an intrinsic
part of the combining site. Though we cannot identify ordered solvent in HuLys at 2.7-Å resolution, it would seem
essential for antigen recognition that CDRs in a humanized
antibody recreate a water network similar to that in the
combining site of the parent mouse antibody.
Received for publication Received for publication 7 October 1997 and in revised form 2 December 1997..
We thank Steve Sheriff, Thomas B. Lavoie, and Greg Winter for suggestions on the manuscript.
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